Synthetic retinoids (in cell modulation)

ABSTRACT

There are described novel compounds of formula I: (I) in which, in which A 1 , A 2 , A 3 , A 4 , R 1  and R 2  are each as herein defined, for use in the treatment or alleviation of an RAR mediated condition; and methods related thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase under 35 U.S.C. § 371 ofInternational Patent Application No. PCT/GB2017/052351 filed on Aug. 9,2017, which claims priority to and benefit of United Kingdom PatentApplication No. 1613712.7 filed on Aug. 9, 2016, and the entirety ofthese applications are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to a novel use of compounds and methods oftreatment related thereto. In one aspect the invention also relates tocertain compounds that are novel per se.

More particularly, the present invention relates to the use of highlyconjugated synthetic retinoid compounds in the generation of reactiveoxygen species and the destruction of cells. The invention also providesa method of medical treatment using the novel compounds of theinvention, for example in a photodynamic therapy.

BACKGROUND TO THE INVENTION

Vitamin A (retinol) and its derivatives belong to a class of compoundsknown as retinoids. Retinoids are an important class of signallingmolecules that are involved in controlling many important biologicalpathways from embryogenesis through to adult homeostasis and manyaspects of stem cell development, such as, stem cell proliferation,differentiation and apoptosis.

Retinoids are structurally and/or functionally related to vitamin A; andmany possess biological activity including all-trans-retinoic acid(ATRA). ATRA is the most abundant endogenous retinoid and has beenwidely studied for many years; ATRA isomerises under physiological andexperimental conditions, with different isomers activating differentreceptors, thus accounting for the variety of biological effectsobserved with these small molecules.

Due to the ability of retinoids to control differentiation and apoptosisin both normal and tumour cells, they have the potential to act aschemopreventative and chemotherapeutic agents, although toxicity hasprevented widespread use.

However, ATRA exhibits poor stability, in particular upon exposure tolight. ATRA compounds isomerise and degrade upon exposure to light. Toovercome this, efforts are made to store and work with ATRA in the dark,but such precautions increase the cost associated with working withATRA, and do not entirely mitigate the problem. Furthermore, as ATRA isliable to photoisomerisation and degradation upon storage, it isdifficult to predict accurately the amount of active compoundadministered in a single dose. Efforts have been made to overcome theproblems associated with ATRA by synthesising stable retinoid compounds.It is generally believed that ATRA is susceptible to photoisomerisationdue to its conjugated linker group.

International Patent application No. PCT/GB2007/003237 (WO 2008/025965)disclosed new retinoid compounds which exhibited good stability andinduced cell differentiation.

Apoptosis is a programmed energy-requiring form of cell death, involvingthe activation of a number of proteases called ‘caspases’. The inductionof apoptosis by ATRA is well documented in the literature. ATRA cantrigger both the induction and suppression of pro-apoptotic genes, aswell as the suppression of several anti-apoptotic genes, leading to anoverall promotion of apoptosis^(1,2) ATRA can induce cell proliferation.However, the pro-apoptotic effect of ATRA is concentration dependent.

Unlike endogenous ATRA, the synthetic retinoids are stable and do notbreak down easily.

Triplet state photosensitizers (PS) usually contain a light-harvestingregion, which is responsible for the dual-functionality oflight-harvesting and intersystem crossing, where electrons in thesinglet state non-radiatively pass to the triplet state. Quenching ofthe triplet-excited state can result in the formation of reactive oxygenspecies (ROS), radicals from ground state molecular oxygen or directchemical reactions with surrounding molecules. High levels of ROS act asa non-selective, highly effective killer of animal, plant, fungal andbacterial cells. Localised ROS production is an immune defence strategyemployed in both animal and plant systems in response to pathogenattack.

However, whilst photoactive compounds are known, most suffer from anumber of drawbacks which make them poor therapeutic agents. Firstly,many exhibit inadequate pharmacological properties, such as poor aqueoussolubility and long biological half-lives, which causes skinphotosensitivity for weeks post-treatment. In addition, many lack, orexhibit poor ability to target specific tissues or cells, oftenresulting in significant off-target damage. Existing photoactivecompounds used for the production of ROS in vivo are also, typically,high molecular weight polymers of varying length, making consistentmanufacture difficult, or contain metals including zinc, palladium,indium, tin or lutetium, which may cause toxicity issues.^(8,9)

International Patent application No. WO 2016/055800 describes a seriesof synthetic, fluorescent small molecules that act as triplet statephotosensitizers when activated by UV or violet (405 nm) light.

The present invention pertains to related synthetic retinoid compoundswith a highly conjugated structure.

Due to the incorporation of an electron donating group, typically anitrogen, the highly conjugated retinoid compounds may be biologicallyinert in the unactivated state, but in the activated state, e.g. byexposure to pulses of low to medium energy short-wavelength visiblelight, can cause the apoptosis of cells.

Importantly, adjacent cells, not exposed to light are unharmed.

SUMMARY TO THE INVENTION

The present invention provides highly conjugated retinoid compoundssuitable for use in, inter alia, photodynamic therapy.

Thus, according to a first aspect of the invention there is provided theuse of a highly conjugated retinoid compound in the generation ofreactive oxygen species when said compound is activated by light.

By the term “highly conjugated retinoid compound” is meant a compoundthat possesses a generally understood retinoid structure that includesat least six conjugated double or triple bonds or equivalents thereof.Such highly conjugated retinoid compound should be understood to possessan increased conjugation when compared to “normal” retinoids i.e. eithernatural retinoic acids or synthetic, direct analogues of retinoic acids,also referred to as synthetic retinoids and arotinoids.

For the avoidance of doubt reference to a retinoid should be construedas including naturally occurring compounds with a retinoid structure,synthetic retinoids and more conjugated homologues thereof. In addition,the term “retinoid” shall include arotinoids (aromatic retinoids).

More specifically, the present invention provides the use as hereindescribed wherein the highly conjugated retinoid compound is a compoundof formula I:

in which

-   -   A¹ is N or CR³;    -   A² is N or CR⁴;    -   A³ is N or CR⁵;    -   A⁴ is N or CR⁶;    -   R³, R⁴, R⁵ and R⁶, which may be the same or different, are each        hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl, aralkyl, halogen,        trifluoroalkyl, cyano, nitro, —NR^(a)R^(b), —OR^(a), glycol,        —C(O)R^(a), —C(O)OR^(a), —OC(O)R^(a), —S(O)R^(a)R^(b),        —C(O)NR^(a)R^(b) or a solubilising group;    -   R¹ is —NR^(7a)R^(7b) or together with R⁶ forms a ring II:

-   -   R⁷ and R^(7a) are each hydrogen, propynyl, —(CH₂)_(n)C≡CH,        —(CH₂)_(n)SH, —(CH₂)SO₂F or —(CH₂)_(n)C═CH₂, alkyl_(C1-10), said        alkyl being optionally substituted by aryl or heteroaryl;    -   R^(7b) is hydrogen, propynyl, alkyl_(C1-10), said alkyl being        optionally substituted by aryl or heteroaryl;    -   R⁸, R⁹, R¹⁰ and R¹¹, which may be the same or different, are        each hydrogen or alkyl_(C1-4), aryl, halogen, trifluoroalkyl,        —OR^(c) or glycol, or together one pair of R⁸ and R¹⁰ or R⁹ and        R¹¹ represent a bond;    -   R¹² and R¹³, which may be the same or different, are each        hydrogen, alkyl_(C1-4) or together one pair of R¹⁰ and R¹² or        R¹¹ and R¹³ represent a bond, or R¹² and R¹³ together form a        group:        ═CR¹⁴R¹⁵    -   provided that the pair of R¹⁰ and R¹² or R¹¹ and R¹³ does not        represent a bond if a pair from R⁸, R⁹, R¹⁰ and R¹¹ represents a        bond;    -   R¹⁴ and R¹⁵, which may be the same or different, are each        hydrogen or alkyl_(C1-10); and    -   R^(a), R^(b) and R^(c), which may be the same or different, are        each hydrogen or alkyl_(C1-10);    -   n is an integer from 1 to 6;    -   R² is a group III:

in which

-   -   X^(a) is —C≡C—, —CH═CH— or N═CH—;    -   X^(b) is —C≡C— or is absent;    -   A⁵ is N or CR¹⁷;    -   A⁶ is N or CR¹⁸;    -   A⁷ is N or CR¹⁹;    -   A⁸ is N or CR²⁰,    -   R¹⁷, R¹⁸, R¹⁹ and R²⁰, which may be the same or different, are        each hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl, aralkyl,        halogen, trifluoroalkyl, cyano, nitro, —NR^(d)R^(e), —OR^(d),        glycol, —C(O)R^(d), —C(O)OR^(d), —OC(O)R^(d), —S(O)R^(d)R^(e),        —C(O)NR^(d)R^(e) or a solubilising group;    -   R¹⁶ is —CR²¹═CR²²Y, —C≡C—R²³ or together with R¹⁸ forms a ring        IV:

-   -   A⁹ is N or CR²⁴;    -   A¹⁰ is N or CR²⁵;    -   A¹¹ is N or CR²⁶;    -   R²³ is a group V:

in which

-   -   A¹² is N or CR²⁷;    -   A¹³ is N or CR²⁸;    -   A¹⁴ is N or CR²⁹;    -   A¹⁵ is N or CR³⁰;    -   R²¹ and R²², which may be the same or different, are each        hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl, halogen or        trifluoroalkyl;    -   R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹ and R³⁰, which may be the same or        different, are each hydrogen, alkyl_(C1-10), alkene-_(C2-12),        aryl, halogen, trifluoroalkyl, —OR^(f), glycol or a solubilising        group;    -   R^(d), R^(e) and R^(f), which may be the same or different, are        each hydrogen or alkyl_(C1-10), Y is —CO₂R³¹, —COH, —CO₂CH₂C≡CH,        —CN, —SF₅, —SO₃H, —SO₂NH₂, —SO₂CF₃, —CF₃, —CO₂(CH₂)_(m)SH,        —CO₂(CH₂)_(m)SO₂F, —CO₂(CH₂)_(m)CH═CH₂, —C═NR³² or —C═N⁺R³³R³⁴;        R³¹ is hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl or a        photocleavable group, such as CH₂aryl-NO₂;    -   R³², R³³ and R³⁴, which may be the same or different, are each        hydrogen, alkyl_(C1-10), alkene_(C2-12) or aryl;    -   m is an integer from 1 to 9;    -   and isomers thereof;        in free or in salt form.

As used herein, the term “alkyl” refers to a fully saturated, branched,unbranched or cyclic hydrocarbon moiety, i.e. primary, secondary ortertiary alkyl or, where appropriate, cycloalkyl or alkyl substituted bycycloalkyl, they may also be saturated or unsaturated alkyl groups.Where not otherwise identified, preferably the alkyl comprises 1 to 10carbon atoms, more preferably 1 to 7 carbon atoms, or 1 to 4 carbonatoms. Representative examples of alkyl include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,n-decyl and the like.

As used herein the term “aryl” refers to an aromatic monocyclic ormulticyclic hydrocarbon ring system consisting only of hydrogen andcarbon and containing from 6 to 19 carbon atoms, preferably 6 to 10carbon atoms, where the ring system may be partially saturated. Arylgroups include, but are not limited to groups such as fluorenyl, phenyl,indenyl and naphthyl. Unless stated otherwise specifically in thespecification, the term “aryl” or the prefix “ar-” (such as in“aralkyl”) is meant to include aryl radicals optionally substituted byone or more substituents selected from the group consisting of alkyl,alkenyl, alkynyl, halo, haloalkyl, cyano, nitro, amino, amidine, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl or heteroarylalkyl. Preferred aryl groups are optionallysubstituted phenyl or naphthyl groups.

An aryl group may be mono-, bi-, tri-, or polycyclic, preferably mono-,bi-, or tricyclic, more preferably mono- or bicyclic.

As used herein, the term “heteroaryl” refers to a 5-14 memberedmonocyclic- or bicyclic- or polycyclic-aromatic ring system, having 1 to8 heteroatoms selected from N, O or S. Preferably, the heteroaryl is a5-10 or 5-7 membered ring system. Typical heteroaryl groups include 2-or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl,3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or5-1,2,4-triazolyl, 4- or 5-1,2, 3-triazolyl, tetrazolyl, 2-, 3-, or4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, 2-,4-, or 5-pyrimidinyl.

The term “heteroaryl” also refers to a group in which a heteroaromaticring is fused to one or more aryl, cycloaliphatic, or heterocyclylrings, where the radical or point of attachment is on the heteroaromaticring. Nonlimiting examples include but are not limited to 1-, 2-, 3-,5-, 6-, 7-, or 8-indolizinyl, 1-, 3-, 4-, 5-, 6-, or 7-isoindolyl, 2-,3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-indazolyl, 2-,4-, 5-, 6-, 7-, or 8-purinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, or9-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl, 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolinyl, 1-, 4-, 5-, 6-, 7-, or 8-phthalazinyl, 2-,3-, 4-, 5-, or 6-naphthyridinyl, 2-, 3-, 5-, 6-, 7-, or 8-quinazolinyl,3-, 4-, 5-, 6-, 7-, or 8-cinnolinyl, 2-, 4-, 6-, or 7-pteridinyl, 1-,2-, 3-, 4-, 5-, 6-, 7-, or 8-4aH carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-,or 8-carbazolyl, 1-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-carbolinyl, 1-, 2-,3-, 4-, 6-, 7-, 8-, 9-, or 10-phenanthridinyl, 1-, 2-, 3-, 4-, 5-, 6-,7-, 8-, or 9-acridinyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-perimidinyl,2-, 3-, 4-, 5-, 6-, 8-, 9-, or 10-phenathrolinyl, 1-, 2-, 3-, 4-, 6-,7-, 8-, or 9-phenazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or10-phenothiazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenoxazinyl,2-, 3-, 4-, 5-, 6-, or 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or10-benzisoqinolinyl, 2-, 3-, 4-, or thieno[2,3-b]furanyl, 2-, 3-, 5-,6-, 7-, 8-, 9-, 10-, or 11-7H-pyrazino[2,3-c]carbazolyl, 2-, 3-, 5-, 6-,or 7-2H-furo[3,2-b]-pyranyl, 2-, 3-, 4-, 5-, 7-, or8-5H-pyrido[2,3-d]-o-oxazinyl, 1-, 3-, or 5-1H-pyrazolo[4,3-d]-oxazolyl,2-, 4-, or 54H-imidazo[4,5-d] thiazolyl, 3-, 5-, or8-pyrazino[2,3-d]pyridazinyl, 2-, 3-, 5-, or 6-imidazo[2,1-b] thiazolyl,1-, 3-, 6-, 7-, 8-, or 9-furo[3,4-c]cinnolinyl, 1-, 2-, 3-, 4-, 5-, 6-,8-, 9-, 10, or 11-4H-pyrido[2,3-c]carbazolyl, 2-, 3-, 6-, or7-imidazo[1,2-b][1,2,4]triazinyl, 7-benzo[b]thienyl, 2-, 4-, 5-, 6-, or7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, 2-, 4-, 4-, 5-, 6-,or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-benzoxapinyl, 2-,4-, 5-, 6-, 7-, or 8-benzoxazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-,or 11-1H-pyrrolo[1,2-b][2]benzazapinyl. Typical fused heteroaryl groupsinclude, but are not limited to 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl,1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 2-, 3-, 4-, 5-, 6-, or7-indolyl, 2-, 3-, 4-, 5-, 6-, 7-benzofuranyl, 2-, 4-, 5-, 6-, or7-benzo[b]thienyl, 2-, 4-, 5-, 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or7-benzimidazolyl, 2-, 4-, 5-, 6-, or 7-benzothiazolyl.

A heteroaryl group may be mono-, bi-, tri-, or polycyclic, preferablymono-, bi-, or tricyclic, more preferably mono- or bicyclic.

A heteroaryl group may optionally be substituted by aryl or aralkyl,e.g. benzyl.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I:

-   -   wherein A¹ is CR³, A² is CR⁴, A³ is CR⁵ and A⁴ is CR⁶; and    -   R¹, R², R³, R⁴, R⁵ and R⁶ are each as herein defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I:

-   -   wherein A¹, A², A³, A⁴ and R² are each as herein defined; and    -   R¹ together with R⁶ forms a ring II:

-   -   wherein R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are each as herein        defined.

Preferably in this aspect of the invention wherein A¹ is CR³, A² is CR⁴,A³ is CR⁵ and A⁴ is CR⁶.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII:

-   -   wherein X^(a) is —C≡C— and X^(b) is —C≡C—; and    -   A⁵, A⁶, A⁷, A⁸ and R¹⁶ are each as herein defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII wherein X^(a) is —C≡C— and X^(b) is absent.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII wherein X^(a) is —CH═CH— and X^(b) is absent.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII wherein X^(a) is N═CH— and X^(b) is absent.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII;

-   -   wherein A⁵ is CR¹⁷, A⁶ is CR¹⁸, A⁷ is CR¹⁹ and A⁸ is CR²⁰, and    -   X^(a), X^(b), R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each as herein        defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII;

-   -   wherein A⁵ is CR¹⁷, A⁶ is CR¹⁸, A⁷ is CR¹⁹ and A⁸ is CR²⁰; and    -   R¹⁷, R¹⁹ and R²⁰ are each as herein defined;    -   R¹⁶ together with R¹⁸ forms a ring IV:

-   -   wherein A⁹, A¹⁰, A¹¹ and Y are each as herein defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII;

-   -   and R¹⁶ is —C≡C—R²³    -   wherein R²³ is a group V:

in which

-   -   A¹² is CR²⁷, A¹³ is CR²⁸, A¹⁴ is CR²⁹ and A¹⁵ is CR³⁰; and    -   R²⁷, R²⁸, R²⁹, R³⁰ and Y are each as herein defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII;

-   -   and R¹⁶ is —C≡C—R²³, R²³ is a group V and Y is CO₂R³¹, —COH,        —CO₂CH₂C≡CH, —CN, —SF₅, —SO₃H, —SO₂NH₂, —SO₂CF₃, in which R³¹ is        as herein defined. Preferably Y is CO₂R³¹ in which R³¹ is as        herein defined. Preferably R³¹ is hydrogen or alkyl_(C1-10).

In one aspect of the invention R⁷ or R^(7a) is alkyl C1-10, preferablyalkyl C1-3.

In one aspect of the invention R⁷ is propynyl, —(CH₂)_(n)C≡CH,—(CH₂)_(n)SH, —(CH₂)_(n)SO₂F or —(CH₂)_(n)C═CH₂; in which n is as hereindefined.

In one aspect of the invention R⁸, R⁹, R¹⁰ and R¹¹ are each hydrogen.

In one aspect of the invention one pair of R⁸ and R¹⁰ or R⁹ and R¹¹represent a bond.

In one aspect of the invention R¹² and R¹³ are the same or different;R¹² and R¹³ may each represent alkyl C1-4, e.g. methyl.

As used herein, the term “halogen” or “halo” refers to fluoro, chloro,bromo, and iodo.

In one aspect of the invention R² is a group VI:

-   -   wherein R³¹ is as herein defined.

In another aspect of the invention R² is a group VII:

-   -   wherein R³¹ is as herein defined.

In another aspect of the invention R² is a group VIII:

-   -   wherein R³¹ is as herein defined.

In another aspect of the invention R² is a group IX:

-   -   wherein R³¹ is as herein defined.

The moiety —CO₂R³¹ is preferably in the 4-position, i.e. in the paraposition to the ethynyl group. Preferably R³¹ is hydrogen.

According to one aspect of the invention there is provided a highlyconjugated retinoid compound of formula IIa:

-   -   in which R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ are each as        herein defined; and    -   R² is a group VI, VII, VIII or IX as herein defined    -   and isomers thereof;        in free or in salt form.

Illustrative compounds of formula I which may be mentioned include thoseselected from the group consisting of:

-   (2E)-3-(4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]    ethynylphenyl)prop-2-enoic acid (DC324);

-   (2E)-3-(4-2-[4,4-dimethyl-1-(propyn-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]    ethynylphenyl)prop-2-enoic acid (DC474); and

-   (2E)-3-(4-2-[4,4-dimethyl-1-(propyn-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]    ethynylphenyl)prop-2-enoic acid methyl ester (DC473);

-   -   and isomers thereof;        in free or in salt form.

Compounds of formula I are either known per se or may be prepared bymethods described in International Patent application No. WO2016/055800.

Excitation of the fluorescent compound to a short-lived excited singletstate occurs upon treatment with light, this light is then emitted asfluorescence when the compound resumes singlet ground state energy.Alternatively, the excited singlet state may instead transfer thisenergy, via intersystem crossing, to an excited triplet state. Thecompound in an excited triplet state can be quenched by a nearby entity,in order to give another compound which is no longer fluorescent,leading to a gradual dimming. It is possible that the compounds of thepresent invention are entering a stable excited triplet state which issusceptible to quenching via a type-I or -II reaction.

In a type-I reaction, the excited triplet state can participate inelectron-transfer to a biological substrate, leading to the formation ofradicals and radical ions. After interaction with oxygen, these radicalscan produce oxygenated products such as superoxide ions, O₂ ⁻. Type-IIreactions generate the short-lived and highly reactive cytotoxic agent,singlet oxygen (¹O₂), by conversion from stable triplet oxygen (³O₂).

Singlet oxygen is much more reactive than triplet oxygen, due to thespin inversion of one of its outer electrons. Rather than two partiallyfilled outer antibonding orbitals each containing one electron of thesame spin, (σ_(2p))²(π_(2px))²(π_(2py))²(π_(2px*))¹(π_(2py*))¹, as isfound in triplet oxygen, singlet oxygen has one filled outer antibondingorbital with the electrons in opposite spin,(σ_(2p))²(π_(2px))²(π_(2py))²(π_(2px*))².

FIG. 1 herein provides a Jablonski diagram showing the formation ofsinglet oxygen. In FIG. 1 PS denotes a photosensitizer in a groundsinglet state. Light energy transforms this in to an excited singletstate, denoted by ¹PS*. Excited singlet states are short lived. They caneither release light energy as fluorescence to return to a singletground state, or form an excited triplet state, denoted by ³PS*. Energytransfer can occur from the excited triplet state to form singletoxygen.³

Human cells are biologically responsive to singlet oxygen production;UVA radiation induces singlet oxygen in cells, which acts as theeffector for a signal transduction pathway dependent on the activationof transcription factor AP-2, a factor also activated by ATRA. Thisleads to the expression of intercellular adhesion molecule-1 (ICAM-1) inhuman epidermal keratinocytes, a protein normally expressed ininflammatory skin diseases.⁴

FIG. 2 herein illustrates the biological effects of ROS generation.Fluorescent compounds excited by light can lose energy in the form ofheat or fluorescence. They can also undergo inter-system crossing andtransition into the triplet state. If the triplet state is long lived itcan generate reactive oxygen species. At higher concentrations ROS canlead to cell death via necrosis or apoptosis.⁵ Notably, in contrast, atlow concentrations, ROS is known to cause cell proliferation.

Irradiation of cells with UV can induce an ROS mediated apoptoticresponse, see FIG. 2 . However, it has also been proven to have aneffect on the retinoid signalling pathway. UV irradiation of humanepidermis in vivo can reduce the expression of RARγ and RXRα at both themRNA and protein levels. This leads to a loss of retinoid responsivegene expression in the skin.⁶ ATRA was found not to enhance UVB-inducedapoptosis, according to a study by Lee et al. Although UVB radiation wasshown to cause significant apoptosis, there was little differencebetween the ATRA treated cells and untreated control.²

Reactive Oxygen Species (ROS) include hydroxyl (·OH), alkoxyl (RO·) orperoxyl (ROO·), superoxide (O·₂) or nitroxyl radicals (NO·), in additionto the non-radicals hydrogen peroxide (H₂O₂), organic hydroperoxides(ROOH) and hypochlorous acid (HOCl). ROS are generated as part of theinflammatory response, and are part of the body's natural defenceagainst microbes, owing to their destructive effect on both proteins andDNA. Under normal conditions, the body's cells are protected against ROSby antioxidants, such as reduced glutathione, catalase, and superoxidedismutase. In addition to their role as destructive agents, ROS alsoplay a part as chemical messengers, involved in receptor-mediatedsignalling pathways and transcriptional activation. ROS are known to beinvolved in the induction of apoptosis, with mitochondria being both thesource and the target of ROS. Oxidation of the mitochondrial pores byROS may disrupt the mitochondrial membrane potential contributing tocytochrome c release.⁷

The light activated compounds of the present invention are especiallysuitable for the manufacture of a medicament for use in photodynamictherapy (PDT).

Thus, according to a further aspect of the invention there is providedthe use of a highly conjugated retinoid compound in the manufacture of amedicament for use in photodynamic therapy (PDT).

According to a yet further aspect of the invention there is provided amethod of treating a patient with photodynamic therapy (PDT), the methodcomprising the administration of a highly conjugated retinoid compoundthat generates reactive oxygen species when said compound is activatedby light.

The compounds of the invention are biologically inert in the unactivatedstate, but short pulses of low to medium energy short-wavelength visiblelight cause defined biological effects. Cells demonstrate a gradedresponse based on the quantity of energy delivered; the lowest energylight induces cells to proliferate in wound healing assays, low tomedium-low energy induces cell apoptosis in labelled cells includingdistinctive membrane blebbing visible within the first 10 minutesfollowing light exposure, and the medium to high energy causes cells todie immediately by necrosis. Importantly, adjacent cells, not exposed tolight are unharmed. Proof of concept experiments have been performed inmammalian cells; however, the ability to generate ROS is not speciesspecific and thus at high concentrations should be effective in killingany ROS sensitive cells, including animal, plant, bacterial and fungalcells.

Thus, according to this aspect of the invention the photodynamic therapy(PDT) may comprise non-surgical cell ablation for the treatment of oneor more of cancer; the treatment of benign growths; the treatment ofimmune mediated inflammatory disorders; or the treatment of a diseasecaused by a pathogenic organism as herein described.

Photodynamic therapy (PDT) can be an effective anticancer treatmentoption. PDT involves the administration of a tumour-localisingphotosensitizer (PS) followed by light activation to generate highlycytotoxic reactive oxygen species (ROS), particularly singlet oxygen,which trigger cell apoptosis and necrosis. By localizing both the PS andthe light exposure to tumour regions, PDT can selectively kill tumourcells while preserving local tissues. PDT has been used to treatpatients with many different types of cancer, including head and necktumours, breast cancer, gynaecological tumours, brain tumours,colorectal cancer, mesothelioma, and pancreatic cancer. The use of PDTfor treating cancers in the head and neck is particularly advantageousover traditional treatment modalities, e.g., surgery and irradiation, asPDT causes less destruction of surrounding tissues and reduces aestheticand functional impairments. The compounds of the present invention canbe used to produce high levels of ROS for the destruction of diseasedtissues, such as cancerous tumours.

Thus, according to one aspect of the invention, the photodynamic therapy(PDT) may comprise the treatment of one or more cancer selected from thegroup wherein the cancer cells are selected from one or more of primarycancer, breast cancer, colon cancer, prostate cancer, non-small celllung cancer, neuroblastoma, glioblastoma, lymphoma, mesothelioma, livercancer, intrahepatic bile duct cancer, oesophageal cancer, pancreaticcancer, stomach cancer, laryngeal cancer, brain cancer, ovarian cancer,testicular cancer, cervical cancer, oral cancer, pharyngeal cancer,renal cancer, thyroid cancer, uterine cancer, urinary bladder cancer,hepatocellular carcinoma, thyroid carcinoma, osteosarcoma, small celllung cancer, leukaemia, myeloma, gastric carcinoma, melanoma, andmetastatic cancers. The compounds of the present invention are suitablefor a photodynamic therapy (PDT) that causes the apoptosis of cancercells.

According to a further aspect of the invention the photodynamic therapy(PDT) may comprise the treatment of benign growths e.g. prostatichyperplasia, keloids or intestinal polyps.

According to a further aspect of the invention the photodynamic therapy(PDT) may comprise the treatment of an immune mediated inflammatorydisease in a mammal, e.g. a human, caused by an aberrant immuneresponse. This may include skin (e.g. psoriasis), joint (e.g. rheumatoidarthritis), intestines (inflammatory bowel diseases) and other localisedand systemic autoimmune diseases; inflammation driven diseasepathologies including graft versus host disease (GVHD) and tissuespecific vasculitis.

According to a further aspect of the invention the photodynamic therapy(PDT) may comprise the treatment of a disease in a mammal, e.g. a human,caused by a pathogenic organism. The pathogenic organism may be onewhich comprises eukaryotic cells or prokaryotic cells. Such pathogenicorganisms include but shall not be limited to, bacteria, viruses, fungi,parasites, protozoa, and toxins as well as cells and tissues infected orinfiltrated therewith.

One objective of the present invention is to provide a photodynamicmethod for inactivation/reduction of bacteria (both Gram-positive andGram-negative) in complex environment like blood, serum and saliva. Afurther object is to provide a therapy suitable for the treatment of aninfectious disease, caused by a pathogenic organism, including but notlimited to bacteria, viruses, fungi, parasites, protozoa, and toxins.

Concentrations of photosensitizer used in the study were as follows: 100μM, 10 μM, 1 μM, 0.1 μM and 0.01 μM.

At low concentrations the compounds of the present invention can deliverlow dose ROS, which can be used to initiate tissue recovery andregeneration through cellular proliferation. Therefore, according afurther aspect of the present invention there is provided a method oftreating a patient with photodynamic therapy (PDT), the methodcomprising the administration of a highly conjugated retinoid compoundthat generates reactive oxygen species when said compound is activatedby light to initiate tissue recovery and/or regeneration.

According to a yet further aspect of the invention chaperones may becovalently bound to the compounds described herein. Such covalentlylinked compound-chaperones can be used for selective targeting of aspecific subset of cells. Suitable chaperones include pharmaceuticalcompounds or biologics, such as, antibodies, e.g. antibodies to cellsurface proteins, cytokines, chemokines, hormones and growth factors(e.g. EGF) that bind to cellular receptors; affimers and other selectivebinding agents, or other transport systems, such as transport proteins,polyglycosides or drug delivery systems, including cyclodextrins etc.

The chaperones may be coupled to the compounds described hereincovalently, for example, by ester or amide linkages. The technique of“click” chemistry, i.e. joining substrates to biomolecules may suitablybe used in the preparation of the compound-chaperones of the presentinvention. One example of a linkage for coupling a compound of theinvention with a chaperone as herein described is a triazolo linkage(see Example 3 herein) which can be attached to the chaperone.

Such compound-chaperones are novel per se. Therefore, according to afurther aspect of the invention there is provided a compound-chaperonecomprising a highly conjugated retinoid as herein described covalentlylinked to a chaperone entity as herein described.

More specifically, the present invention provides a compound-chaperonecomprising a compound of formula I as herein defined covalently linkedto a chaperone entity.

According to this aspect of the invention there is especially provided acompound-chaperone wherein the chaperone is a biologic as hereindefined.

The invention further provides the use of a compound-chaperone in thegeneration of reactive oxygen species when said compound is activated bylight.

As described herein, the compounds of the invention are biologicallyinert in the unactivated state. Furthermore, the compounds of theinvention, including the compound-chaperone entities described hereinare advantageous in that they are, inter alia, bioavailable, or morebioavailable than conventionally known compounds used in photodynamictherapy. In addition, conventionally known compounds used inphotodynamic therapy comprise an organometallic agent. Thus, thecompounds described herein including the compound-chaperone entities arealso advantageous in that they are, inter alia, non-metallic and aregenerally less toxic than known compounds used in photodynamic therapy.

In addition, the invention provides a method of treating a patient withphotodynamic therapy (PDT), the method comprising the administration ofa compound-chaperone that generates reactive oxygen species when saidcompound is activated by light.

Furthermore, certain of the compounds of formula I are also novel perse. Thus, according to this aspect of the invention there is provided ahighly conjugated retinoid compound is a compound of formula I:

in which

-   -   A¹ is N or CR³;    -   A² is N or CR⁴;    -   A³ is N or CR⁵;    -   A⁴ is N or CR⁶;    -   R³, R⁴, R⁵ and R⁶, which may be the same or different, are each        hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl, aralkyl, halogen,        trifluoroalkyl, cyano, nitro, —NR^(a)R^(b), —OR^(a), glycol,        —C(O)R^(a), —C(O)OR^(a), —OC(O)R^(a), —S(O)R^(a)R^(b),        —C(O)NR^(a)R^(b) or a solubilising group;    -   R¹ is —NR^(7a)R^(7b) or together with R⁶ forms a ring II:

-   -   R⁷ and R^(7a) are each hydrogen, propynyl, —(CH₂)_(n)C≡CH,        —(CH₂)_(n)SH, —(CH₂)_(n)SO₂F or —(CH₂)_(n)C═CH₂, alkyl_(C1-10),        said alkyl being optionally substituted by aryl or heteroaryl;    -   R^(7b) is hydrogen, propynyl, alkyl_(C1-10), said alkyl being        optionally substituted by aryl or heteroaryl;    -   R⁸, R⁹, R¹⁰ and R¹¹, which may be the same or different, are        each hydrogen or alkyl_(C1-4), aryl, halogen, trifluoroalkyl,        —OR^(c) or glycol, or together one pair of R⁸ and R¹⁰ or R⁹ and        R¹¹ represent a bond;    -   R¹² and R¹³, which may be the same or different, are each        hydrogen, alkyl_(C1-4) or together one pair of R¹⁰ and R¹² or        R¹¹ and R¹³ represent a bond, or R¹² and R¹³ together form a        group:        ═CR¹⁴R¹⁵    -   provided that the pair of R¹⁰ and R¹² or R¹¹ and R¹³ does not        represent a bond if a pair from R⁸, R⁹, R¹⁰ and R¹¹ represents a        bond;    -   R¹⁴ and R¹⁵, which may be the same or different, are each        hydrogen or alkyl_(C1-10); and    -   R^(a), R^(b) and R^(c), which may be the same or different, are        each hydrogen or alkyl_(C1-10);    -   n is an integer from 1 to 6;    -   R² is a group III:

in which

-   -   X^(a) is —C≡C—, —CH═CH— or N═CH—;    -   X^(b) is —C≡C— or is absent;    -   A⁵ is N or CR¹⁷;    -   A⁶ is N or CR¹⁸;    -   A⁷ is N or CR¹⁹;    -   A⁸ is N or CR²⁰;    -   R¹⁷, R¹⁸, R¹⁹ and R²⁰, which may be the same or different, are        each hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl, aralkyl,        halogen, trifluoroalkyl, cyano, nitro, —NR^(d)R^(e), —OR^(d),        glycol, —C(O)R^(d), —C(O)OR^(d), —OC(O)R^(d), —S(O)R^(d)R^(e),        —C(O)NR^(d)R^(e) or a solubilising group;    -   R¹⁶ is —CR²¹═CR²²Y, —C≡C—R²³ or together with R¹⁸ forms a ring        IV:

-   -   A⁹ is N or CR²⁴;    -   A¹⁰ is N or CR²⁵;    -   A¹¹ is N or CR²⁶;    -   R²³ is a group V:

in which

-   -   A¹² is N or CR²⁷;    -   A¹³ is N or CR²⁸;    -   A¹⁴ is N or CR²⁹;    -   A¹⁵ is N or CR³⁰;    -   R²¹ and R²², which may be the same or different, are each        hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl, halogen or        trifluoroalkyl;    -   R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹ and R³⁰, which may be the same or        different, are each hydrogen, alkyl_(C1-10), alkene-_(C2-12),        aryl, halogen, trifluoroalkyl, —OR^(f), glycol or a solubilising        group;    -   R^(d), R^(e) and R^(f), which may be the same or different, are        each hydrogen or alkyl_(C1-10);    -   Y is —CO₂R³¹, —COH, —CO₂CH₂C≡CH, —CN, —SF_(S), —SO₃H, —SO₂NH₂,        —SO₂CF₃, —CF₃, —CO₂(CH₂)_(m)SH, —CO₂(CH₂)_(m)SO₂F,        —CO₂(CH₂)_(m)CH═CH₂, —C═NR³² or C═N⁺R³³R³⁴; R³¹ is hydrogen,        alkyl_(C1-10), alkene_(C2-12), aryl or a photocleavable group,        such as CH₂aryl-NO₂;    -   R³², R³³ and R³⁴, which may be the same or different, are each        hydrogen, alkyl_(C1-10), alkene_(C2-12) or aryl;    -   m is an integer from 1 to 9;    -   and isomers thereof;        in free or in salt form.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein described, provided that whenR⁷ is methyl, R⁸, R⁹, R¹⁰ and R¹¹ are each hydrogen, X^(a) is —C≡C— andX^(b) is absent, Y is not —CO₂R³¹.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I:

-   -   wherein A¹ is CR³, A² is CR⁴, A³ is CR⁵ and A⁴ is CR⁶; and    -   R¹, R², R³, R⁴, R⁵ and R⁶ are each as herein defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I:

-   -   wherein A¹, A², A³, A⁴ and R² are each as herein defined; and    -   R¹ together with R⁶ forms a ring II:

-   -   wherein R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are each as herein        defined.

Preferably in this aspect of the invention wherein A¹ is CR³, A² is CR⁴,A³ is CR⁵ and A⁴ is CR⁶.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII:

-   -   wherein X^(a) is —C≡C— and X^(b) is —C≡C—; and    -   A⁵, A⁶, A⁷, A⁸ and R¹⁶ are each as herein defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII wherein X^(a) is —C≡C— and X^(b) is absent.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII wherein X^(a) is —CH═CH— and X^(b) is absent.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII wherein X^(a) is —N═CH— and X^(b) is absent.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII;

-   -   wherein A⁵ is CR¹⁷, A⁶ is CR¹⁸, A⁷ is CR¹⁹ and A⁸ is CR²⁰, and    -   X^(a), X^(b), R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each as herein        defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII;

-   -   wherein A⁵ is CR¹⁷, A⁶ is CR¹⁸, A⁷ is CR¹⁹ and A⁸ is CR²⁰; and    -   R¹⁷, R¹⁹ and R²⁰ are each as herein defined;    -   R¹⁶ together with R¹⁸ forms a ring IV:

-   -   wherein A⁹, A¹⁰, A¹¹ and Y are each as herein defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII;

-   -   and R¹⁶ is —C≡C—R²³    -   wherein R²³ is a group V:

in which

-   -   A¹² is CR²⁷, A¹³ is CR²⁸, A¹⁴ is CR²⁹ and A¹⁵ is CR³⁰; and    -   R²⁷, R²⁸, R²⁹, R³⁰ and Y are each as herein defined.

In one aspect of the invention there is provided a highly conjugatedretinoid compound of formula I as herein defined wherein R² is a groupIII;

-   -   and R¹⁶ is —C≡C—R²³, R²³ is a group V and Y is —CO₂R³¹, —COH,        —CO₂CH₂C≡CH, —CN, —SF₅, —SO₃H, —SO₂NH₂, —SO₂CF₃, in which R³¹ is        as herein defined. Preferably Y is —CO₂R³¹ in which R³¹ is as        herein defined. Preferably R³¹ is hydrogen or alkyl_(C1-10).

In one aspect of the invention R⁷ or R^(7a) is alkyl C1-10, preferablyalkyl C1-3.

In one aspect of the invention R⁸, R⁹, R¹⁰ and R¹¹ are each hydrogen.

In one aspect of the invention one pair of R⁸ and R¹⁰ or R⁹ and R¹¹represent a bond.

In one aspect of the invention R¹² and R¹³ are the same or different;R¹² and R¹³ may each represent alkyl C1-4, e.g. methyl.

As used herein, the term “halogen” or “halo” refers to fluoro, chloro,bromo, and iodo.

In one aspect of the invention R² is a group VI:

-   -   wherein R³¹ is as herein defined.

In another aspect of the invention R² is a group VII:

-   -   wherein R³¹ is as herein defined.

In another aspect of the invention R² is a group VIII:

-   -   wherein R³¹ is as herein defined.

In another aspect of the invention R² is a group IX:

-   -   wherein R³¹ is as herein defined.

The moiety —CO₂R³¹ is preferably in the 4-position, i.e. in the paraposition to the ethynyl group. Preferably R³¹ is hydrogen.

Novel compounds which may be specifically mentioned include:

-   (2E)-3-(4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoic    acid (DC324);

-   (2E)-3-(4-2-[4,4-dimethyl-1-(propyn-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoic    acid (DC474); and

-   (2E)-3-(4-2-[4,4-dimethyl-1-(propyn-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoic    acid methyl ester (DC473);

-   -   and isomers thereof;        in free or in salt form.

The novel compounds of the invention may be prepared by methods knownper se. The novel compounds of formula I may be prepared using methodsknown to the person skilled in the art or by methods described herein.Examples of such preparations are shown schematically:

Generally, the wavelength suitable for use in the use or the method ofthe present invention may depend on several factors, such as the depthof the targeted disease site, and the structure or property of thehighly conjugated retinoid compound. Thus, the wavelength suitable foractivating the highly conjugated retinoid compound may desirably be inthe UV spectrum or the blue/violet range of the visible spectrum. Moreespecially the wavelength suitable for activating the highly conjugatedretinoid compound may be from about 10 to about 750 nm; preferably fromabout 100 to about 650 nm; more preferably from about 250 to about 450nm. The use of a laser diode operating at a wavelength of 405 nm isparticularly useful.

According to a further aspect of the present invention there is provideda composition comprising one or more of the compounds of the presentinvention in combination with one or more pharmaceutically acceptableexcipients for use in the generation of reactive oxygen species whensaid compound is activated by light as herein described.

Thus, according to this aspect of the invention there is provided acomposition as herein described for use in the treatment of one or moreof cancer; the treatment of benign growths; the treatment of immunemediated inflammatory disorders; or a disease caused by a pathogenicorganism as herein described.

The composition of the present invention also includes one or morepharmaceutically acceptable carriers, excipients, adjuvants or diluents.The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings or, as the case may be, an animalwithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

The invention further provides a composition as herein described for usein the treatment of one or more of cancer; the treatment of benigngrowths; the treatment of immune mediated inflammatory disorders; or adisease caused by a pathogenic organism as herein described. Thecomposition of the invention may comprise the compounds described hereinor the compound-chaperone entities described herein.

When the composition of the invention is prepared for oraladministration, the compounds described above are generally combinedwith a pharmaceutically acceptable carrier, diluent or excipient to forma pharmaceutical formulation, or unit dosage form.

For oral administration, the composition may be in the form of a powder,a granular formation, a solution, a suspension, an emulsion or in anatural or synthetic polymer or resin for ingestion of the activeingredients from a chewing gum. The composition may also be presented asa bolus, electuary or paste. Orally administered compositions of theinvention can also be formulated for sustained release, e.g. thecompounds described above can be coated, microencapsulated, or otherwiseplaced within a sustained delivery device. The total active ingredientsin such formulations comprise from 0.1 to 99.9% by weight of theformulation.

Thus, one or more suitable unit dosage forms comprising the compounds ofthe invention can be administered by a variety of routes including oral,parenteral (including subcutaneous, intravenous, intramuscular andintraperitoneal), rectal, dermal, transdermal, intrathoracic,intrapulmonary, mucosal, intraocular and intranasal (respiratory)routes. The composition may also be formulated in a lipid formulation orfor sustained release, for example, using microencapsulation. Theformulations may, where appropriate, be conveniently presented indiscrete unit dosage forms and may be prepared by any of the methodswell known to the pharmaceutical arts. Such methods may include the stepof mixing the therapeutic agent with liquid carriers, solid matrices,semi-solid carriers, finely divided solid carriers or combinationsthereof, and then, if necessary, introducing or shaping the product intothe desired delivery system.

Pharmaceutical formulations comprising the compounds of the inventioncan be prepared by procedures known in the art using well-known andreadily available ingredients. For example, the compound can beformulated with common excipients, diluents, or carriers, and formedinto tablets, capsules, solutions, suspensions, powders, aerosols andthe like. Examples of excipients, diluents, and carriers that aresuitable for such formulations include buffers, as well as fillers andextenders such as starch, cellulose, sugars, mannitol, and silicicderivatives.

Binding agents can also be included such as carboxymethyl cellulose,hydroxymethylcellulose, hydroxypropyl methylcellulose and othercellulose derivatives, alginates, gelatine, and polyvinylpyrrolidone.Moisturising agents can be included such as glycerol, disintegratingagents such as calcium carbonate and sodium bicarbonate. Agents forretarding dissolution can also be included such as paraffin. Resorptionaccelerators such as quaternary ammonium compounds can also be included.Surface active agents such as cetyl alcohol and glycerol monostearatecan be included. Adsorptive carriers such as kaolin and bentonite can beadded. Lubricants such as talc, calcium and magnesium stearate, andsolid polyethyl glycols can also be included. Preservatives may also beadded. The compositions of the invention can also contain thickeningagents such as cellulose and/or cellulose derivatives. They may alsocontain gums such as xanthan, guar or carbo gum or gum arabic, oralternatively polyethylene glycols, bentones and montmorillonites, andthe like.

For example, tablets or caplets containing the compounds of theinvention can include buffering agents such as calcium carbonate,magnesium oxide and magnesium carbonate. Suitable buffering agents mayalso include acetic acid in a salt, citric acid in a salt, boric acid ina salt and phosphoric acid in a salt. Caplets and tablets can alsoinclude inactive ingredients such as cellulose, pregelatinised starch,silicon dioxide, hydroxyl propyl methyl cellulose, magnesium stearate,microcrystalline cellulose, starch, talc, titanium dioxide, benzoicacid, citric acid, corn starch, mineral oil, polypropylene glycol,sodium phosphate, zinc stearate, and the like. Hard or soft gelatinecapsules containing at least one compound of the invention can containinactive ingredients such as gelatine, microcrystalline cellulose,sodium lauryl sulphate, starch, talc, and titanium dioxide, and thelike, as well as liquid vehicles such as polyethylene glycols (PEGs) andvegetable oil. Moreover, enteric-coated caplets or tablets containingone or more compounds of the invention are designed to resistdisintegration in the stomach and dissolve in the more neutral toalkaline environment of the duodenum.

The therapeutic compounds of the invention can also be formulated aselixirs or solutions for convenient oral administration or as solutionsappropriate for parenteral administration, for instance byintramuscular, subcutaneous, intraperitoneal or intravenous routes. Thepharmaceutical formulations of the therapeutic compounds of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension orsalve.

Thus, the therapeutic compounds may be formulated for parenteraladministration (e.g. by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers. As noted above, preservatives can be added to help maintainthe shelve life of the dosage form. The active compound(s) and otheringredients may form suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activecompound(s) and other ingredients may be in powder form, obtained byaseptic isolation of sterile solid or by lyophilisation from solutionfor constitution with a suitable vehicle, e.g., sterile, pyrogen-freewater before use.

It is possible to add, if necessary, an adjuvant chosen fromantioxidants, surfactants, other preservatives, film-forming,keratolytic or comedolytic agents, perfumes, flavourings and colourings.Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole,butylated hydroxytoluene and α-tocopherol and its derivatives can beadded.

These formulations can contain pharmaceutically acceptable carriers,vehicles and adjuvants that are well known in the art. It is possible,for example, to prepare solutions using one or more organic solvent(s)that is/are acceptable from the physiological standpoint, chosen, inaddition to water, from solvents such as acetone, acetic acid, ethanol,isopropyl alcohol, dimethyl sulphoxide, glycol ethers such as theproducts sold under the name “Dowanol”, polyglycols and polyethyleneglycols, C1-C4 alkyl esters of short-chain acids, ethyl or isopropyllactate, fatty acid triglycerides such as the products marketed underthe name “Miglyol”, isopropyl myristate, animal, mineral and vegetableoils and polysiloxanes.

Preferably, the composition is in the form of a solvent or diluentcomprising one or more of the compounds as described above. Solvents ordiluents may include acid solutions, dimethylsulphone,N-(2-mercaptopropionyl) glycine, 2-n-nonyl-1,3-dioxolane and ethylalcohol. Preferably the solvent/diluent is an acidic solvent, forexample, acetic acid, citric acid, boric acid, lactic acid, propionicacid, phosphoric acid, benzoic acid, butyric acid, malic acid, malonicacid, oxalic acid, succinic acid or tartaric acid.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that areavailable in the art. Examples of such substances include normal salinesolutions such as physiologically buffered saline solutions and water.Specific non-limiting examples of the carriers and/or diluents that areuseful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable buffered saline solutionssuch as phosphate buffered saline solutions pH 7.0-8.0.

The solvent may comprise an acetic acid solution. The solvent, forexample acetic acid solution, may be present in the composition at aconcentration of less than 1%, 0.5%, 0.25%, 0.1%, 0.05% or 0.01% w/wacid, for example acetic acid.

The composition of the present invention may comprise one or moreadditional therapeutic agents. For instance, where the composition ofthe present invention is useful in the treatment or prevention ofcancer, one or more additional chemotherapeutic and or chemopreventativeagents may be included.

When the composition of the present invention is for use in thetreatment or prevention of cancer, the one or more additionalchemotherapeutic and or chemopreventative agents may be selected fromthe group consisting of: a chemotherapeutic agent, an immunotherapeuticagent, a gene therapy agent, and a radiotherapeutic agent.

According to this aspect of the invention the composition of the presentinvention may be administered in combination, separately, simultaneouslyor sequentially, with a second therapy wherein the second therapy isselected from the group consisting of one or more of a chemotherapeuticagent; an alkylating agent, such as carmustine or temozolamide; amitotic inhibitor, such as taxanes, (e.g. paclitaxol or docetaxol) orvinca alkaloids (e.g. vinblastine, vincristine, vindestine orvinorelbine); platinum derived compounds (e.g. carboplatin, cisplatin,nedaplatin, oxaliplatin, triplatin tetranitrate or satraplatin);dihydrofolate reductase inhibitors (e.g. aminopterin, methotrexate,pemetrexed or pralatrexate); a DNA polymerase inhibitor (e.g.cytarabine); a ribonucleotide reductase inhibitor (e.g. gemcitabine); athymidylate synthase inhibitors (e.g. fluorouracil, capecitabine,tegafur, carmofur or floxuridine); aspirin; a non-steroidalanti-inflammatory agent (e.g. ibuprofen); a steroidal anti-inflammatoryagent (e.g. a corticosteroid, such as, prednisolone or cortisol); anon-drug oncology therapeutic agent; radiotherapy; tumour embolisation;surgery; and ultrasound.

Where the composition is useful in the treatment of a disease caused bya pathogenic organism, e.g. a bacterial infection and fungal infection;one or more additional antibacterial agents or antifungal agents may beused.

Additionally, the compounds of the present invention are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activecompound, for example, in a particular part of the intestinal orrespiratory tract, possibly over a period of time. Coatings, envelopes,and protective matrices may be made, for example, from polymericsubstances, such as polylactide-glycolates, liposomes, microemulsions,microparticles, nanoparticles, or waxes. These coatings, envelopes, andprotective matrices are useful to coat indwelling devices, e.g. stents,catheters, peritoneal dialysis tubing, draining devices and the like.

For topical administration, the active agents may be formulated as isknown in the art for direct application to a target area. Forms chieflyconditioned for topical application take the form, for example, ofcreams, milks, gels, powders, dispersion or microemulsions, lotionsthickened to a greater or lesser extent, impregnated pads, ointments orsticks, aerosol formulations (e.g. sprays or foams), soaps, detergents,lotions or cakes of soap. Other conventional forms for this purposeinclude wound dressings, coated bandages or other polymer coverings,ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus,the therapeutic compounds of the invention can be delivered via patchesor bandages for dermal administration. Alternatively, the therapeuticcompounds can be formulated to be part of an adhesive polymer, such aspolyacrylate or acrylate/vinyl acetate copolymer. For long-termapplications it might be desirable to use microporous and/or breathablebacking laminates, so hydration or maceration of the skin can beminimized. The backing layer can be any appropriate thickness that willprovide the desired protective and support functions. A suitablethickness will generally be from about 10 to about 200 μm.

Pharmaceutical formulations for topical administration may comprise, forexample, a physiologically acceptable buffered saline solutioncontaining between about 0.001 mg/ml and about 100 mg/ml, for examplebetween 0.1 mg/ml and 10 mg/ml, of one or more of the compounds of thepresent invention specific for the indication or disease to be treated.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcolouring agents. The active compounds can also be delivered viaiontophoresis. The percentage by weight of a therapeutic agent of theinvention present in a topical formulation will depend on variousfactors, but generally will be from about 0.01% to 95% of the totalweight of the formulation, and typically about 0.1-85% by weight.

Drops, such as eye drops or nose drops, may be formulated with one ormore of the therapeutic compounds in an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents orsuspending agents. Liquid sprays can be pumped, or are convenientlydelivered from pressurized packs. Drops can be delivered via a simpleeye dropper-capped bottle, via a plastic bottle adapted to deliverliquid contents drop-wise, or via a specially shaped closure.

The therapeutic compound may further be formulated for topicaladministration in the mouth or throat. For example, the activeingredients may be formulated as a lozenge further comprising aflavoured base, usually sucrose and acacia or tragacanth; pastillescomprising the composition in an inert base such as gelatine andglycerine or sucrose and acacia; and mouthwashes comprising thecomposition of the present invention in a suitable liquid carrier.

The compounds of the invention can also be administered to therespiratory tract. Thus, the present invention also provides aerosolpharmaceutical formulations and dosage forms for use in the methods ofthe invention. In general, such dosage forms comprise an amount of atleast one of the agents of the invention effective to treat or preventthe clinical symptoms of a specific infection, indication or disease.Any statistically significant attenuation of one or more symptoms of aninfection, indication or disease that has been treated pursuant to themethod of the present invention is considered to be a treatment of suchinfection, indication or disease within the scope of the invention.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof the therapeutic agent and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form in,for example, capsules or cartridges, or, e.g. gelatine or blister packsfrom which the powder may be administered with the aid of an inhalator,insufflator, or a metered-dose inhaler.

The compounds of the present invention can also be administered in anaqueous solution when administered in an aerosol or inhaled form. Thus,other aerosol pharmaceutical formulations may comprise, for example, aphysiologically acceptable buffered saline solution containing betweenabout 0.001 mg/ml and about 100 mg/ml of one or more of the compounds ofthe present invention specific for the indication or disease to betreated. Dry aerosol in the form of finely divided solid particles ofthe compounds described above that are not dissolved or suspended in aliquid are also useful in the practice of the present invention.Compounds of the present invention may be formulated as dusting powdersand comprise finely divided particles having an average particle size ofbetween about 1 and 5 μm, alternatively between 2 and 3 μm. Finelydivided particles may be prepared by pulverization and screen filtrationusing techniques well-known in the art. The particles may beadministered by inhaling a predetermined quantity of the finely dividedmaterial, which can be in the form of a powder. It will be appreciatedthat the unit content of active ingredient or ingredients contained inan individual aerosol dose of each dosage form need not in itselfconstitute an effective amount for treating the particular infection,indication or disease since the necessary effective amount can bereached by administration of a plurality of dosage units. Moreover, theeffective amount may be achieved using less than the dose in the dosageform, either individually, or in a series of administrations.

For administration to the upper (nasal) or lower respiratory tract byinhalation, the therapeutic compounds of the invention are convenientlydelivered from a nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

The compounds or compound-chaperones of the present invention may beadministered direct to the eye of a patient, e.g. by injection for thetreatment or prevention of ocular disorders, such as age-related maculardegeneration (AMD). In such treatments the compound orcompound-chaperone may be administered to the eye followed by excitationin a photodynamic therapy.

Furthermore, the active ingredients may also be used in combination withother therapeutic agents, for example one or more of pain relievers,anti-inflammatory agents, antihistamines, bronchodilators,chemoprotective agents, chemotherapeutic agents, antibacterial agentsand the like.

According to a yet further aspect of the invention there is provided amethod of generating a reactive oxygen species which comprises the lightactivation of a highly conjugated retinoid compound as herein described.

According to an additional aspect of the invention there is provided aprocess for the manufacture of a compound of formula I as hereindescribed which comprises reacting a compound of formula X;

-   -   in which A¹, A², A³, A⁴ and R¹ are each as herein defined; and    -   Z¹ is a leaving group, for example, halogen, pseudohalogen,        boronic acid or boronate ester;        with a compound of formula XI;        R²H  XI    -   in which R² is as herein defined.

Alternatively, a process for the manufacture of a compound of formula Ias herein described may comprise reacting a compound of formula XII;

-   -   in which A¹, A², A³, A⁴ and R¹ are each as herein defined;        with a compound of formula XIII:        R²Z¹  XIII    -   in which Z¹ is a leaving group, for example, halogen,        pseudohalogen, boronic acid or boronate ester.

Compounds of formula XII may be prepared by dealkylation of a compoundof formula I in which R² is alkyl as herein described.

Compounds of formula I may be prepared using methods known to the personskilled in the art or by methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described by way of example only withreference to the accompanying figures in which:

FIG. 1 is a Jablonski diagram showing the formation of singlet oxygen;

FIG. 2 illustrates the biological effects of ROS generation;

FIG. 3 illustrates DC324 localised to one side of the nucleus in largepatches of cells;

FIG. 4 illustrates DC324 co-imaged with BODIPY® Golgi stain;

FIG. 5 illustrates that DC324 was the most rapid inducer of cell death;

FIG. 6 illustrates that cell death observations were consistent acrossdifferent cell lines;

FIG. 7 illustrates that the majority of DMSO treated cells appearedhealthy after 47 exposures to 405 nm laser, at 50% strength;

FIG. 8 illustrates that the majority of ATRA treated cells appearedhealthy after 47 exposures to 405 nm laser, at 50% strength;

FIG. 9 illustrates that membrane blebbing was induced in DC324 treatedcells by 405 nm laser, at 50% strength;

FIG. 10 illustrates DC324 treated cells putatively producing apoptoticbodies;

FIG. 11 illustrates DC324 fluorescence from treated HaCaT cells, imagedwith 405 nm laser light, at 50% strength;

FIG. 12 illustrates the dose-dependent response of HaCaT cells treatedwith DC324 and DC473 and 10 seconds of UV;

FIG. 13 illustrates control treatments stained for superoxide afterexposure to UV;

FIG. 14 illustrates active compounds stained for superoxide afterexposure to DAPI;

FIG. 15 illustrates inactive DC324 compound treated cells stained forsuperoxide after UV exposure;

FIG. 16 illustrates DC473 compound treated cells after 2 hours and 24hours UV exposure;

FIG. 17 illustrates a comparison of DC324 and DC473 compound treatedcells;

FIG. 18 illustrates the combined normalised absorbance spectra of DC324,DC473 and DC474 in CHCl₃ (10 μM);

FIG. 19 illustrates the combined normalised emission spectra of DC324,DC473 and DC474 in CHCl₃ (100 nM) with excitation at their respectivemaximal absorption wavelengths;

FIG. 20 illustrates the combined normalised absorbance spectra of DC473,DC474 and click-conjugate compound 16 in CHCl₃ (10 μM); and

FIG. 21 illustrates the combined normalised emission spectra of DC473,DC474, and click-conjugate compound 16 in CHCl₃ (100 nM) with excitationat their respective maximal absorption wavelengths.

In the figures, any reference to DC271 is a reference to compound 9 ofExample 3 of WO 2016/055800. Reference to DC324 is a reference tocompound 9 of Example 1 herein; and reference to DC473 is a reference tocompound 14 of Example 2.5 herein; reference to DC474 is a reference tocompound 15 of Example 2.6 herein.

The following abbreviations are used in the Examples and other parts ofthe description:

-   -   ATRA: All Trans-Retinoic Acid    -   DCM: dichloromethane    -   DMF: N,N-dimethylformamide    -   DMSO: dimethylsulfoxideEDTA: ethylenediaminetetraacetic acid    -   EtOAc: ethyl acetate    -   GCMS: gas chromatography-mass spectrometry    -   h: hour(s)    -   KOAc: potassium acetate    -   RT: room temperature    -   THF: tetrahydrofuran        General Experimental

Reagents were purchased from Sigma-Aldrich, Acros Organics, Alfa-Aesarand Fluorochem and used without further purification unless otherwisestated. Solvents were used as supplied, and dried before use withappropriate drying agents if stated. Reactions were monitored in situ byTLC, or NMR spectroscopy. Thin layer chromatography (TLC) was conductedusing Merck Millipore silica gel 60G F254 25 glassplates withvisualisation by UV lamp. Flash column chromatography was performedusing SiO₂ from Sigma-Aldrich (230-400 mesh, 40-63 μm, 60 Å) andmonitored using TLC. NMR spectra were recorded on Varian VNMRS-700,Varian VNMRS-600, Bruker Avance-400 or Varian Mercury-400 spectrometersoperating at ambient probe temperature unless otherwise stated. NMRspectra were recorded in CDCl₃ or DMSO-d₆ purchased from GossScientific. NMR peaks are reported as singlet (s), doublet (d), triplet(t), quartet (q), broad (br), heptet (hept), combinations thereof, or asa multiplet (m). ES-MS was performed by the Durham Universitydepartmental service using a TQD (Waters UK) mass spectrometer andAcquity UPLC (Waters Ltd, UK), and accurate mass measurements wereobtained using a QTOF Premier mass spectrometer and an Acquity UPLC(Waters Ltd, UK). GCMS was performed by the Durham Universitydepartmental service using a Shimadzu QP2010-Ultra. IR spectra wererecorded on a Perkin Elmer FT-IR spectrometer. Melting points wereobtained using a Gallenkamp melting point apparatus. Elemental analyseswere obtained by the Durham University departmental service using anExeter Analytical CE-440 analyzer.

Synthetic Procedures

EXAMPLE 1(2E)-3-(4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoicacid (9) 1.1 N-(4-Iodophenyl)-3-methylbut-2-enamide (1)

To a solution of 4-iodoaniline (25.0 g, 114.0 mmol) in DCM (400 mL) wasadded 3,3-dimethylacryloyl chloride (13.36 mL, 120.0 mmol) and theresultant white suspension was stirred for 0.5 h, after which pyridine(9.70 mL, 120 mmol) was added and the solution stirred at RT for 16 h.The solution was diluted with DCM and H₂O, washed with sat. NH₄Cl, H₂Oand brine, dried (MgSO₄) and evaporated to give a crude light brownsolid (33 g) which was recrystallised from EtOH to give compound 1 as awhite crystalline solid (31.8 g, 93%): m.p.=136-138° C.; ¹H NMR (700MHz, CDCl₃) δ 1.91 (s, 3H), 2.22 (s, 3H), 5.68 (s, 1H), 7.01 (s, 1H),7.33 (m, 2H), 7.60 (d, J=8.8 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 20.2,27.6, 87.2, 118.7, 122.0, 137.8, 138.2, 154.1, 165.5; IR (neat)ν_(max)/cm⁻¹ 3294m, 3094, 2964w, 2890w, 1666m, 1586m, 1430m, 821s, 650m;MS (ES): m/z=302.0 [M+H]⁺; HRMS (ES) calcd. for C₁₁H₁₃NOI [M+H]⁺:302.0042, found: 302.0050; Found: C, 43.87; H, 4.02; N, 4.64. Calc. forC₁₁H₁₂NOI: C, 43.88; H, 4.02; N, 4.65%.

1.2 6-Iodo-4,4-dimethyl-1,2,3,4-tetrahydroquinolin-2-one (2)

Compound 1 (11.5 g, 38.3 mmol) and AlCl₃ (7.66 g, 57.5 mmol) were addedto anhydrous DCM (150 mL) under Ar and the resultant solution stirredvigorously for 2.5 h at RT. The reaction was cooled to 0° C., quenchedslowly with H₂O, diluted with DCM, stirred with 5% NaOH (w/v) until thesolution turned off-white, then further washed with H₂O and brine, dried(MgSO₄) and evaporated to give a crude yellow solid (12.0 g). This wasrecrystallised from EtOH to give compound 2 as a white crystalline solid(10.2 g, 88%): m.p.=199-202° C.; ¹H NMR (700 MHz, CDCl₃) δ 1.32 (s, 6H),2.47 (s, 2H), 6.62 (d, J=8.3 Hz, 1H), 7.47 (dd, J=8.3, 1.9 Hz, 1H), 7.56(d, J=1.8 Hz, 1H), 9.20 (s, 1H); ¹³C NMR (176 MHz, CDCl₃) δ 27.7, 34.2,45.2, 86.8, 118.1, 133.7, 135.1, 135.9, 136.6, 171.3; IR (neat)ν_(max)/cm⁻¹ 3164m, 3102, 3040w, 2953m, 1671s, 1596m, 1484m, 817s; MS(ES): m/z=302.0 [M+H]⁺; HRMS (ES) calcd. for C₁₁H₁₃NOI [M+H]⁺: 302.0042,found: 302.0042; Found: C, 43.91; H, 4.02; N, 4.63. Calc. for C₁₁H₁₂NOI:C, 43.88; H, 4.02; N, 4.65%.

1.3 6-Iodo-4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-2-one(3)

To a solution of compound 2 (25.9 g, 85.9 mmol) in anhydrous DMF (200mL) was added crushed KOH (14.5 g, 257 mmol) and the resultant slurrystirred for 1 h at 50° C. under Ar. To this was added 2-iodopropane(25.6 mL, 257 mmol) and the solution stirred at 50° C. for 40 h underAr. The reaction was quenched with H₂O, diluted with EtOAc, washed withsat. NH₄Cl, H₂O and brine, dried (MgSO₄) and evaporated to give a crudeclear oil (29.0 g). This was purified by SiO₂ chromatography(hexane:EtOAc, 9:1, with 1% Et₃N, as eluent) to give compound 3 as acolourless oil (14.8 g, 50%): R_(f) 0.51 (hexane:EtOAc, 8:2, with 1%Et₃N); ¹H NMR (400 MHz, CDCl₃) δ 1.25 (s, 6H), 1.50 (d, J=7.0 Hz, 6H),2.38 (s, 2H), 4.66 (sept, J=7.0 Hz, 1H), 6.87 (d, J=8.6 Hz, 1H), 7.50(dd, J=8.6, 2.1 Hz, 1H), 7.52 (d, J=2.1 Hz, 1H); ¹³C NMR (101 MHz,CDCl₃) δ 20.3, 26.8, 33.1, 47.2, 48.8, 86.9, 119.0, 133.4, 135.9, 139.1,139.3, 169.8; IR (neat) ν_(max)/cm⁻¹ 2961m, 2934w, 2870w, 1667s, 1582m,1482m, 809s; MS (ES): m/z=344.0 [M+H]⁺; HRMS (ES) calcd. for C₁₄H₁₉NOI[M+H]⁺: 344.0511, found: 344.0512; Found: C, 49.21; H, 5.29; N, 4.08.Calc. for C₁₄H₁₈NOI: C, 48.99; H, 5.29; N, 4.08%.

1.4 6-Iodo-4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinoline (4)

To a solution of compound 3 (1.25 g, 3.63 mmol) in anhydrous toluene (15mL) was added borane dimethyl sulfide complex (2.0 M in THF, 1.91 mL,3.81 mmol) dropwise and the resultant solution stirred at reflux for 16h under Ar. The solution was cooled to RT, 10% aq. Na₂CO₃ (25 ml) addedand then stirred for 0.5 h. The resultant solution was diluted withEtOAc, washed with H₂O and brine, dried (MgSO₄) and evaporated to give acrude colourless oil (1.12 g). This was purified by SiO₂ chromatography(hexane:EtOAc, 9:1, with 1% Et₃N, as eluent) to give compound 4 as acolourless oil (1.08 g, 90%): ¹NMR (700 MHz, CDCl₃) δ 1.19 (d, J=6.6 Hz,6H), 1.24 (s, 6H), 1.65-1.67 (m, 2H), 3.14-3.17 (m, 2H), 4.06 (sept,J=6.6 Hz, 1H), 6.46 (d, J=8.8 Hz, 1H), 7.28 (dd, J=8.9, 2.1 Hz, 1H),7.39 (d, J=2.2 Hz, 1H); ¹³C NMR (176 MHz, CDCl₃) δ 18.9, 30.3, 32.4,36.6, 36.8, 47.3, 76.1, 113.4, 134.5, 134.8, 135.6, 144.0; IR (neat)ν_(max)/cm⁻¹ 2957m, 2927w, 2863w, 1580m, 1489m, 792s, 684w; MS (ES):m/z=330.1 [M+H]⁺; HRMS (ES) calcd. for C₁₄H₂₁NI [M+H]⁺: 330.0719, found:330.0717.

1.5 4-Iodobenzenediazonium tetrafluoroborate (5)

4-Iodoaniline (10.95 g, 50 mmol) was added to tetrafluoroboric acidsolution (48% in H₂O, 25 mL), and the suspension was cooled to 0° C.before a solution of NaNO₂ (3.79 g, 55 mmol) in H₂O (13.73 mL) was addeddropwise with vigorous stirring so as to maintain the internaltemperature below 5° C. After addition the suspension was furtherstirred for 1 h at 0° C., before the precipitated solid was isolated byfiltration, washed with cold MeOH and dried to give a crude brown solid.This was dissolved in a minimal amount of acetone (around 55 mL), and towhich Et₂O was slowly added to precipitate a yellow solid. This wasfiltered, washed with cold Et₂O and dried to give compound 5 as a paleyellow solid (13.13 g, 83%): ¹H NMR (700 MHz, (CD₃)₂SO) δ 8.35 (d, J=9.0Hz, 2H), 8.43 (d, J=9.0 Hz, 2H); ¹³C NMR (151 MHz, (CD₃)₂SO) δ 113.6,115.1, 132.8, 140.2; IR (neat) ν_(max)/cm⁻¹ 3090 w, 2282 s, 1548 m, 1461w, 824 s, 523 m; Found: C, 22.83; H, 1.30; N 8.83, Calc. for C₆H₄BF₄IN₂:C, 22.67; H, 1.27; N, 8.81%.

1.6 Methyl (2E)-3-(4-iodophenyl)prop-2-enoate (6)

Pd(OAc)₂ (0.138 g, 0.61 mmol), CaCO₃ (2.40 g, 24.0 mmol) and compound 5(5.54 g, 17.4 mmol) were suspended in MeOH (60 mL). Methyl acrylate(2.16 mL, 24.0 mmol) was added, and the suspension was stirredvigorously for 1.5 h. The solution was diluted with DCM, filteredthrough Celite and evaporated to give a crude light brown solid (6.3 g).This was purified by SiO₂ chromatography (hexane:DCM, 1:1, as eluent) togive compound 6 as a white solid (3.74 g, 75%): ¹NMR (600 MHz, CDCl₃) δ3.81 (s, 3H), 6.44 (d, J=16.0 Hz, 1H), 7.24 (d, J=8.4 Hz, 2H), 7.60 (d,J=16.0 Hz, 1H), 7.73 (d, J=8.4 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃) δ 52.0,96.7, 118.8, 129.7, 134.1, 138.3, 143.8, 167.3; IR (neat) ν_(max)/cm⁻¹3080w, 3000w, 2850w, 1708s, 1636m, 1580m, 1483m, 815 s, 493 m; MS (ES):m/z=288.9 [M+H]⁺; FIRMS (ES) calcd. for C₁₀H₁₀IO₂ [M+H]⁺: 288.9726,found: 288.9733; Found: C, 41.86; H, 3.14. Calc. for C₁₀H₉IO₂: C, 41.96;H, 3.15%.

1.7 Methyl (2E)-3-4-[2-(trimethylsilyl)ethynyl]phenylprop-2-enoate (7)

Triethylamine (80 mL) was added to an oven-dried Schlenk flask, and wasthen degassed via sonication under vacuum, followed by refilling with Ar(×5). Pd(PPh₃)₂Cl₂ (0.217 g, 0.31 mmol), CuI (0.06 g, 0.31 mmol) andcompound 6 (3.57 g, 12.38 mmol) and trimethylsilylacetylene (1.76 mL,12.44 mmol) were then added and the mixture was stirred at RT overnight.The solution was diluted with Et₂O, passed through Celite/SiO₂ undervacuum, and evaporated to give a light brown solid (4.5 g). This waspurified by SiO₂ chromatography (hexane:EtOAc, 9:1, as eluent) to givecompound 7 as a white solid (2.65 g, 83%): ¹H NMR (400 MHz, CDCl₃) δ0.25 (s, 9H) 3.80 (s, 3H), 6.42 (d, J=16.0 Hz, 1H), 7.40-7.50 (m, 4H),7.64 (d, J=16.0 Hz, 1H).

1.8 Methyl (2E)-3-(4-ethynylphenyl)prop-2-enoate (8)

Compound 7 (2.21 g, 8.55 mmol) was dissolved in THF (25 mL), and cooledto −20° C. Tetrabutylammonium fluoride (1.0 M in THF, 8.98 mL, 8.98mmol) was then added dropwise and the resultant solution stirred at −20°C. for 1 h, after which H₂O was added, and the solution extracted withEtOAc (3×). The organics were washed with brine, dried (MgSO₄) andevaporated to give a crude brown solid. This was purified by SiO₂chromatography (hexane:EtOAc, 9:1, as eluent) to give compound 8 as awhite solid (1.52 g, 95%): m.p.=93-95° C.; ¹H NMR (600 MHz; CDCl₃) δ3.18 (s, 1H), 3.81 (s, 3H), 6.44 (d, J=16.0 Hz, 1H), 7.46-7.51 (m, 4H),7.66 (d, J=16.0 Hz, 1H); ¹³C NMR (151 MHz; CDCl₃) δ 52.0, 79.4, 83.3,119.1, 124.2, 128.1, 132.8, 134.9, 143.9, 167.4; IR (neat) ν_(max)/cm⁻¹3260m, 2996w, 2946w, 2108w, 1700s, 1634m, 1554m, 1431m, 1206s, 831s; MS(EI): m/z=186.1 [M]⁺; Found: C, 77.40; H, 5.37. Calc. for C₁₂H₁₀O₂: C,77.40; H, 5.41%.

1.9(2E)-3-(4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoic acid (9) (DC324)

Compound 4 (0.61 g, 1.85 mmol) was dissolved in triethylamine (12 mL),and the resultant solution was degassed by sonication under vacuum,before the atmosphere was replaced with Ar (5×). Pd(PPh₃)₂Cl₂ (0.13 g,0.185 mmol), CuI (0.0352 g, 0.185 mmol) and compound 8 (0.362 g, 1.94mmol) were then added under Ar. The resultant suspension was stirred atRT for 72 h. The suspension was diluted with hexane and passed through athin Celite/SiO₂ plug (eluting with hexane, then hexane:EtOAc (8:2)).The extracts were washed with sat. NH₄Cl (3×), brine, dried (MgSO₄) andevaporated to give the coupling product as an orange solid (0.7 g). Thiswas dissolved in THF (20 mL), 20% NaOH (2 mL) added, and the resultantsolution was stirred under reflux for 40 h. The mixture was cooled,acidified to pH 1 with 5% HCl, diluted with EtOAc, washed with sat.NaHCO₃, H₂O and brine, dried (MgSO₄) and evaporated to give a crudeyellow solid which was recrystallised from MeOH to give compound 9 as anorange crystalline solid (0.46 g, 67% over two steps): ¹H NMR (700 MHz;(CD₃)₂SO) δ 1.16 (d, J=6.6 Hz, 6H)), 1.22 (s, 6H), 1.60-1.65 (m, 2H),3.16-3.21 (m, 2H), 4.14 (hept, J=6.6 Hz, 1H), 6.54 (d, J=16.0 Hz, 1H),6.69 (d, J=9.4 Hz, 1H), 7.17 (dd, J=8.6, 2.1 Hz, 1H), 7.29 (d, J=2.2 Hz,1H), 7.46-7.51 (m, 2H), 7.58 (d, J=16.0 Hz, 1H), 7.66-7.72 (m, 2H),12.41 (s, 1H); ¹³C NMR (176 MHz; (CD₃)₂SO) δ 18.6, 29.7, 31.6, 35.9,36.1, 46.6, 86.9, 94.0, 106.8, 110.5, 119.5, 125.2, 128.4, 128.8, 130.6,131.1, 131.2, 133.3, 143.0, 144.5, 167.5; MS(ES): m/z=374.2 [M+H]⁺;FIRMS (ES) calcd. for C₂₅H₂₈NO₂ [M+H]⁺: 374.2120, found 374.2118.

EXAMPLE 2(2E)-3-(4-{2-[4,4-Dimethyl-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl}phenyl)prop-2-enoicacid (15) (DC474) 2.1 (3-Bromoprop-1-yn-1-yl)trimethylsilane (10)

A solution of propargyl bromide (80% in toluene, 6.69 mL, 60.0 mmol) inTHF (75 mL) was cooled to −78° C. under Ar. Lithiumbis(trimethylsilyl)amide (10.37 g, 62.0 mmol) was added under Ar, andthe solution then stirred for 0.5 h. Chlorotrimethylsilane (10.15 mL,80.0 mmol) was then added dropwise, and the solution stirred for 0.5 h,whereupon sat. NH₄Cl (30 mL) was added, and the solution then warmed toRT. The solution was diluted with EtOAc, washed with brine, dried(MgSO₄) and evaporated to give a crude oil. This was purified by SiO₂chromatography (hexane as eluent), and then further purified byKugelrohr distillation (70-90° C., ambient pressure) to give compound 10as a clear oil (8.09 g, 70%): ¹H NMR (400 MHz; CDCl₃) δ 0.18 (s, 9H),3.91 (s, 2H); ¹³C NMR (101 MHz; CDCl₃) δ −0.1, 14.9, 92.5, 100.2; IR(neat) ν_(max)/cm⁻¹ 2960w, 2906w, 2180w, 1251m, 1204m, 1038m, 837s;MS(EI): m/z=174.9 [M−CH₃]⁺.

2.2 6-Iodo-4,4-dimethyl-1,2,3,4-tetrahydroquinoline (11)

To a solution of compound 2 (4.00 g, 13.28 mmol) in anhydrous toluene(30 mL) was added borane dimethyl sulfide complex (2.0 M in THF, 8.30mL, 16.6 mmol) dropwise and the resultant solution stirred under refluxfor 16 h. The solution was cooled to RT, 10% aq. Na₂CO₃ (25 ml) addedand the solution stirred for 0.5 h. The solution was then diluted withEtOAc, washed with brine, dried (MgSO₄) and evaporated to give a crudered oil. This was purified by SiO₂ chromatography (hexane:EtOAc, 9:1,with 1% Et₃N, as eluent) to give compound 11 as a colourless oil (3.36g, 88%): ¹H NMR (700 MHz; CDCl₃) δ 1.27 (s, 6H), 1.68-1.71 (m, 2H),3.28-3.32 (m, 2H), 3.93 (br, 1H), 6.24 (d, J=8.4 Hz, 1H), 7.19 (dd,J=8.4, 2.0 Hz, 1H), 7.41 (d, J=2.1 Hz, 1H); ¹³C NMR (176 MHz; CDCl₃) δ30.9, 32.0, 36.8, 38.4, 77.7, 116.5, 133.1, 135.1, 135.3, 143.4; IR(neat) ν_(max)/cm⁻¹340br, 2956w, 2927w, 2862w, 1589m, 1524m, 1492s,1352m, 1282s, 804 s; MS(ES): m/z=288.0 [M+H]⁺; HRMS (ES) calcd. forC₁₁H₁₅NI [M+H]⁺: 288.0246, found 288.0242.

2.36-Iodo-4,4-dimethyl-1-[3-(trimethylsilyl)prop-2-yn-1-yl]-1,2,3,4-tetrahydroquinoline(12)

K₂CO₃ (1.39 g, 10.08 mmol) was added to a solution of compound 11 (2.07g, 7.20 mmol) in anhydrous DMF (25 mL) under Ar and the resultant slurrywas stirred for 1 h. Compound 10 (1.65 mL, 10.08 mmol) was added, andthe solution was stirred at RT for 72 h. The solution was diluted withH₂O, and extracted with EtOAc (3×). The organics were washed with sat.NH₄Cl, H₂O and brine, dried (MgSO₄) and evaporated to give a crudeyellow oil. This was purified by SiO₂ chromatography (hexane:EtOAc,96:4, with 1% Et₃N as eluent) to give compound 12 as a light yellow oil(2.71 g, 95%): ¹H NMR (700 MHz; CDCl₃) δ 0.13 (s, 9H), 1.26 (s, 6H),1.74-1.78 (m, 2H), 3.27-3.31 (m, 2H), 3.99 (s, 2H), 6.49 (d, J=8.7 Hz,1H), 7.33 (dd, J=8.7, 2.2 Hz, 1H), 7.42 (d, J=2.2 Hz, 1H); ¹³C NMR (176MHz, CDCl₃) δ 0.2, 30.7, 32.4, 36.9, 42.0, 45.6, 78.8, 89.0, 101.4,114.7, 134.6, 135.5, 135.5, 143.3; IR (neat) ν_(max)/cm⁻¹ 2958w, 2925w,2856w, 2169w, 1584m, 1491m, 1332m, 1248m, 838s; MS(ES): m/z=288.0[M+H]⁺; HRMS (ES) calcd. for C₁₇H₂₅SiNI [M+H]⁺: 398.0801, found398.0797.

2.4 Methyl(2E)-3-[4-(2-{4,4-dimethyl-1-[3-(trimethylsilyl)prop-2-yn-1-yl]-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl)phenyl}prop-2-enoate(13)

Compound 12 (1.61 g, 4.05 mmol) was dissolved in triethylamine (35 mL),and the resultant solution was degassed by sonication under vacuum,before the atmosphere was replaced with Ar (5×). Pd(PPh₃)₂Cl₂ (0.28 g,0.405 mmol), CuI (0.077 g, 0.405 mmol) and compound 8 (0.79 g, 4.25mmol) were then added under Ar. The resultant suspension was stirred atRT for 72 h. The suspension was diluted with hexane and passed through athin Celite/SiO₂ plug (eluting with hexane, then Et₂O). The extractswere washed with sat. NH₄Cl (3×), brine, dried (MgSO₄) and evaporated togive the coupling a crude yellow oil. This was purified by SiO₂chromatography (hexane:EtOAc, 9:1, with 1% Et₃N as eluent) to givecompound 13 as a thick yellow oil (1.25 g, 68%): ¹H NMR (700 MHz, CDCl₃)δ 0.12 (s, 9H), 1.30 (s, 6H), 1.77-1.80 (m, 2H), 3.34-3.37 (m, 2H), 3.81(s, 3H), 4.05 (s, 2H), 6.43 (d, J=16.0 Hz, 1H), 6.68 (d, J=8.6 Hz, 1H),7.27 (dd, J=8.5, 2.0 Hz, 1H), 7.38 (d, J=2.0 Hz, 1H), 7.45-7.52 (m, 4H),7.67 (d, J=16.0 Hz, 1H); ¹³C NMR (176 MHz, CDCl₃) δ 0.2, 30.5, 32.3,36.9, 42.0, 45.7, 51.9, 87.3, 89.0, 93.8, 101.3, 110.5, 112.2, 118.0,126.5, 128.2, 129.6, 130.8, 131.8, 132.5, 133.4, 144.1, 144.4, 167.6; IR(neat) ν_(max)/cm⁻¹ 3042w, 2957w, 2927w, 2858w, 2195w, 1718s, 1634m,1595s, 1515s, 1324s, 1170s, 842s; MS(ES): m/z=456.2 [M+H]⁺; HRMS (ES)calcd. for C₂₆H₂₆NO₂ [M+H]⁺: 456.2359, found 456.2345.

2.5 Methyl(2E)-3-(4-{2-[4,4-dimethyl-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl}phenyl)prop-2-enoate(14) (DC473)

Compound 13 (1.20 g, 2.64 mmol) was dissolved in THF (30 mL), and cooledto −20° C. Tetrabutylammonium fluoride (1.0 M in THF, 2.90 mL, 2.90mmol) was then added dropwise and the resultant solution stirred at −20°C. for 1 h, after which H₂O was added, and the solution extracted withEtOAc (3×). The organics were washed with brine, dried (MgSO₄) andevaporated to give a crude solid. This was purified by SiO₂chromatography (hexane:EtOAc, 8:2, with 1% Et₃N as eluent) to givecompound 14 as a yellow oil that slowly crystallises to give an orangesolid (0.83 g, 82%): m.p.=101-102° C.; ¹H NMR (400 MHz, CDCl₃) δ 1.30(s, 6H), 1.76-1.83 (m, 2H), 2.16-2.20 (m, 1H), 3.33-3.39 (m, 2H), 3.81(s, 3H), 4.05 (d, J=2.4 Hz, 2H), 6.43 (d, J=16.0 Hz, 1H), 6.68 (d, J=8.6Hz, 1H), 7.28 (dd, J=8.5, 1.9 Hz, 1H), 7.39 (d, J=1.9 Hz, 1H), 7.43-7.54(m, 4H), 7.67 (d, J=16.0 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 30.7, 32.3,36.9, 41.0, 45.9, 51.9, 72.0, 79.3, 87.3, 93.7, 110.9, 111.9, 118.0,126.5, 128.2, 129.8, 130.8, 131.9, 132.6, 133.5, 143.9, 144.4, 167.6; IR(neat) ν_(max)/cm⁻¹ 3288w, 2954w, 2927w, 2861w, 2194m, 1716s, 1633m,1595s, 1515s, 1496m, 1324s, 1170s, 830s; MS(ES): m/z=384.4 [M+H]⁺; HRMS(ES) calcd. for C₂₆H₂₆NO₂ [M+H]⁺: 384.1964, found 384.1963.

2.6(2E)-3-(4-{2-[4,4-Dimethyl-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl}phenyl)prop-2-enoicacid (15) (DC474)

Compound 14 (0.824 g, 2.15 mmol) was dissolved in THF (25 mL), 20% NaOH(2.5 mL) added, and the resultant solution was stirred under reflux for40 h. The mixture was cooled, acidified to pH 1 with 5% HCl, dilutedwith EtOAc, washed with sat. NH₄Cl, H₂O and brine, dried (MgSO₄) andevaporated to give a crude yellow solid which was recrystallised fromMeCN to give compound 15 as an orange crystalline solid (0.50 g, 63%):m.p.=193-195° C. (decomposition); ¹H NMR (400 MHz, (CD₃)₂SO) δ 1.24 (s,6H), 1.69-1.75 (m, 2H), 3.12 (t, J=2.3 Hz, 1H), 3.27-3.32 (m, 2H), 4.14(d, J=2.3 Hz, 2H), 6.55 (d, J=16.0 Hz, 1H), 6.74 (d, J=8.7 Hz, 1H), 7.23(dd, J=8.5, 2.0 Hz, 1H), 7.35 (d, J=2.1 Hz, 1H), 7.49-7.53 (m, 2H), 7.59(d, J=16.0 Hz, 1H), 7.68-7.72 (m, 2H), 12.44 (s, 1H); ¹³C NMR (101 MHz,(CD₃)₂SO) δ 30.3, 31.6, 36.0, 44.8, 74.3, 79.8, 87.1, 93.4, 109.2,112.2, 119.7, 124.9, 128.4, 129.1, 130.2, 131.2, 132.1, 133.5, 143.0,143.9, 167.5; IR (neat) ν_(max)/cm⁻¹ 3278w, 2962w, 2920w, 2847w, 2196w,1684.9, 1623m, 1515m, 1217s, 836w; MS(ES): m/z=370.8 [M+H]⁺; HRMS (ES)calcd. for C₂₅H₂₄NO₂ [M+H]⁺: 370.1807, found 370.1804.

EXAMPLE 3 3.1 Example Click Conjugation of Compound 15 (DC474) withBenzyl Azide to Give methyl(2E)-3-[4-(2-{1-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-4,4-dimethyl-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl)phenyl}prop-2-enoate(16)

Compound 15 (75 mg, 0.203 mmol) and benzyl azide (0.028 mL, 0.223 mmol)were suspended in H₂O/^(t)BuOH (1:1, 1 mL), before sodium ascorbate (1Min H₂O, 0.020 mL, 0.02 mmol) and CuSO₄·5H₂O (5 mg, 0.02 mmol) wereadded. The suspension was stirred vigorously for 16 h, before beingdiluted with cold H₂O, and the precipitated solid was filtered and driedto give compound 16 as a yellow solid (59 mg, 57%). ¹H NMR indicated 87%conversion to compound 16.

EXAMPLE 4 4.1 Cell Culture and Media

Immortalised HaCaT cell lines were maintained in Dulbecco's ModifiedEagle's Medium (5 mL) containing 10% Foetal Bovine Serum and 1%Penicillin Streptomycin antibiotic.

4.2 Fixed Cell Imaging

HaCaT human keratinocyte cells were seeded onto acid washed glasscoverslips and treated, with a single dose, of either a 1 μM or a 10 μMsolution of the compound. They were left in this compound containingmedia for 2 to 72 h before live-staining with Mitotracker red or BODIPY®TR Ceramide complexed to BSA. The cells were then fixed with 4% PFA, andmounted onto glass slides with Mowiol®. Images were taken on a Zeiss LSM880 microscope. Note: these experiments were also carried out using amouse embryonic fibroblast cell line, J2. However, some backgroundfluorescence was present in J2s at the same wavelengths as the compound.Therefore, HaCaTs were favoured for imaging.

Treatment with Compounds

An acid washed glass coverslip was placed in the bottom of each well ofa 24 well plate. In each well, HaCaT cells (12,000 approx.) were seededin serum containing media (1 mL) and allowed 24 h to settle before beingtreated. The media was replaced with media containing the compound/DMSOcontrol (1 mL). For the 10 μM wells, compound (10 μL, 1 mM) was added toserum containing media (9990 μL). For the 1 μM wells, compound (1 μL, 1mM) was added to serum containing media (9999 μL). The cells werestained and fixed 72 h or 48 h after being treated with the compound.The appropriate vehicle only experimental controls were included.

MitoTracker® red staining: The cells were incubated for 30 min withMitoTracker® Red diluted in serum containing media (1 mL, 0.1 μM). Theywere then rinsed twice with PBS before incubation with PFA (0.5 mL, 4%),for 5 min. Cells were rinsed in PBS before mounting onto glass slideswith Mowiol®.

BODIPY® TR Ceramide complexed to BSA staining: The cells were incubatedfor 30 min at 4° C. with BODIPY® TR Ceramide complexed to BSA diluted inPBS (1 mL, 5 μM). They were then rinsed twice with cold PBS beforeincubation in fresh PBS at 37° C. for a further 30 min. The cells werefixed by incubation with PFA (0.5 mL, 4%), for 5 min. Cells were rinsedin PBS before mounting onto glass slides with Mowiol®.

Imaging: Images taken using the Zeiss LSM 880 AxioObserver confocalmicroscope, with the Plan-Apochromat 63×/1.4 Oil DIC M27 objective lens.Acquisition settings can be found in table 1.

TABLE 1 Acquisition Settings Green Channel (Compound) Red Channel(Stain) Detection wavelength/nm 431-560 600-735 Excitation wavelength/nm405 594 Emission wavelength/nm 460 668

TABLE 1 Absorption and fluorescence emission maxima of dyes, in nm,determined in methanol BODIPY ® TR Ceramide complexed to BSAMitoTracker ® Red Absorbance wavelength/nm 589 max 579 max 300-640 range300-630 range Emission wavelength/nm 617 max 599 max 575-724 range560-700 range

TABLE 3 Absorbance and emission spectra data DC324 DC473 DC474 (Inchloroform, (In chloroform, (In chloroform, excitation excitationexcitation at 400 nm) at 380 nm) at 300 nm) Absorbance 400 max 380 max380 max wavelength/ 275-475 range 275-450 range 275-450 range nmEmission 560 max 520 max 540 max wavelength/ 450-700 range 440-680 range450-700 range nm

EXAMPLE 5

Living Cell Imaging

5.1 Cell Death on Zeiss Live Cell Observer

One day prior to imaging, HaCaT or J2 cells (20,000 approx.) were seededinto each well of a 6 well or 24 well plastic plate. Cells were allowedto incubate overnight in serum containing media. One to four hoursbefore imaging the normal media was replaced with compound containingmedia, 1 mL per well. The cells were imaged in a 37° C. heated chamberand supplied with 5% CO₂. Zeiss Live Cell Observer microscope: OSRAM1×HBO 103 W/2 100 Watt Mercury Bulb, DAPI Excitation filter=335-385 nm,DAPI Emission filter=420-470 nm. Note: Cells were imaged in DMEM mediawhich contains phenol red.

5.2 Cell Death on Zeiss 880 Confocal

One day prior to imaging, HaCaT cells (4000 approx.) were seeded intoeach well of an 8 well glass slide. Cells were allowed to incubateovernight in serum containing media. One hour before imaging the normalmedia was replaced with compound containing media, 1 mL per well. Thecells were imaged in a 37° C. heated chamber using the Zeiss LSM 880with Airyscan confocal laser scanning microscope. Note: Cells wereimaged in DMEM media which contains phenol red.

5.3 ROS Staining

One day prior to imaging, HaCaT cells (20,000 approx) were seeded intoeach well of a 24 well plastic plate. Cells were allowed to incubateovernight in serum containing media. Three hours prior to imaging, themedia in each well was replaced with fresh serum containing media whichincluded a 1:500 dilution of Superoxide Detection Reagent, CellRox.Note: A 1:2500 dilution was originally tried and was less effective. Thecells were incubated with the ROS stain at 37° C. for one hour. An hourand a half before imaging, the media in the negative control well wasreplaced with N-acetyl-L-cysteine diluted in serum containing media (1mL, 10 mM). One hour before imaging the media in all but one of theother wells was replaced with compound containing media, 1 mL per well.Thirty minutes prior to imaging, the media in the positive control wellwas replaced with pyocyanin diluted in serum containing media (1 mL, 500μM). Pyocyanin induces ROS within 20-30 minutes. The cells were imagedin a heated chamber and supplied with 5% CO₂. The cells were imaged in a37° C. heated chamber using the Zeiss LSM 880 with Airyscan confocallaser scanning microscope. Cells were irradiated with 405 nm laser toactivate compounds and ROS dye was excited with 633 nm laser Note: Cellswere imaged in DMEM media which contains phenol red.

6. Results 6.1 Initial Screening of Compounds

Following exposure to UV light, no membrane blebbing was observed inATRA, EC23 and DMSO treated cells. DC324 and DC473 induced membraneblebbing and cell death following exposure to UV light.

6.2 DC324

DC324 was not observed filling the nucleus.

Referring to FIG. 3 : DC324 localised to one side of the nucleus inlarge patches of cells. DC324 emits above zero fluorescence in the405-640 range. The red channel was recorded 600-735 nm. This image showssigns of DC324 emission in the red channel.

Referring to FIG. 4 : DC324 co-imaged with BODIPY® Golgi stain. Someregions show overlap between the Golgi and DC324. DC324 appears to beentering the nucleus in one of these cells. Cells fixed after 72 hoursin 1 μM DC324.

6.3 Cell Death 6.3.1 UV Light

Referring to FIG. 5 : DC324 was the most rapid inducer of cell death. Asingle 10 s treatment with UV light was sufficient to induce necrosis in10 μM DC324 treated cells, within two hours using a 10× objective lensand at Phase 0. Part C shows healthy cells from the same well, in anarea away from the site of UV exposure. The localised effect of the UVlight indicates that the cell death is not due to heat transfer.

Referring to FIG. 6 : Cell death observations were consistent acrossdifferent cell lines. DC324 treated J2 cells, a mouse fibroblast cellline, displayed membrane blebbing within two hours following a single UVtreatment. UV untreated cells appear healthy after 26 hours in thecompound in the same well as the treated area. This suggests that thecause of death is localised, not due to heat transfer, and due to thecombined effect of the UV light and the DC324. A 10× objective lens atPhase 0 was used.

6.3.2 405 nm Light

Images of 10 μM compound treated cells were taken every twenty minutesand at each time point a treatment of 405 nm light at 50% laser strengthwas applied. Cell death was not as rapid under the 405 nm laser light asunder the UV light, only DC324 was significantly different to the DMSOcontrol.

Referring to FIG. 7 : The majority of DMSO treated cells appearedhealthy after 47 exposures to 405 nm laser, at 50% strength. No membraneblebbing was observed.

Referring to FIG. 8 : The majority of ATRA treated cells appearedhealthy after 47 exposures to 405 nm laser, at 50% strength. No membraneblebbing was observed.

Referring to FIG. 9 : Membrane blebbing was induced in DC324 treatedcells by 405 nm laser, at 50% strength. The first bleb appeared after 21exposures, however, the majority of cells presented membrane blebbingbetween 30 and 40 exposures. The cell death induced by 405 nm lightappeared more characteristic of apoptosis than the cell death caused byUV light. Cells appeared to shrink rather than expand, and apoptoticbodies are visible.

Referring to FIG. 10 : DC324 treated cells putatively producingapoptotic bodies. This is an image from the same experiment discussedpreviously. It was taken at the 37^(th) exposure to 405 nm light, 740minutes from the first exposure, and 770 minutes after the addition ofDC324. A number of the cells appear to be releasing small spheres fromall edges.

Referring to FIG. 11 : DC324 fluorescence from treated HaCaT cells,imaged with 405 nm laser light, at 50% strength. Initially, DC324 isbrightest around the edges of the cells. DC324 is then concentrated tothe centre of the cells and is also visible in the membrane blebs.

6.4 Dose Response

The speed of death was tested at a number of concentrations in order todetermine an estimate for the EC₅₀ (half maximal effectiveconcentration) value. Morphological changes were recorded using timelapse imaging at 5 minute intervals. The time taken between 10 s of UVlight treatment and the first sign of damage to the cell membranes wasrecorded. Each cell in the field of view was treated as an individualdata point, before taking an average of the time for each concentration.

Referring to FIG. 12 : The dose-dependent response of HaCaT cellstreated with DC324 and DC473 and 10 seconds of UV. UV exposure wasadministered through the DAPI filter and a 20× objective lens, phase 0.EC₅₀ values for both compounds were determined based on the averageviable fraction of the cellular population 24 hours after the initialirradiation event of 0.20 (±0.007) μM and 0.18 (±0.006) μM for DC324 andDC473, respectively.

6.5 ROS Staining

Referring to FIG. 13 : Control treatments stained for superoxide afterexposure to UV. Staining for superoxide production under the rhodaminefilter. Fluorescence in the negative control indicates possiblebackground fluorescence from HaCaT cells. One cell in the negativecontrol is induced to produce increased fluorescence post-UV imaging.This could be due to an increase in superoxide production in anapoptotic cell, or merely due to the change in shape of the cell as itdies. The positive control treated cells were visibly rounded and dyingwhen imaged under phase. Therefore, the reactive oxygen species hadalready been produced before the imaging began. However, the superoxidestain was brighter for the positive control, indicating that superoxidewas still present. Several smaller patches of the DMSO treated cellsshowed superoxide production prior to exposure to UV, these cells weremost affected by the DAPI treatment. Compared with the initial picturetaken 60 seconds after UV treatment, the image taken 260 seconds afterUV treatment shows that there is an increase in fluorescence in adefined circle in the centre of these cells.

Referring to FIG. 14 : Active compounds stained for superoxide afterexposure to DAPI. As seen with the DMSO control, the ATRA treated cellswhich already had some evidence of superoxide production before UVtreatment produced an increased level of fluorescence post exposure toUV. As with the DMSO control, this fluorescence formed a defined circlein the centre of the cell, 260 seconds after exposure to UV. For alltreatments, healthy normal cells produced a brighter fluorescence insmall circles in the cell.

Referring to FIG. 15 : DC324 compound treated cells stained forsuperoxide after UV exposure. The intensity of the small bright circlesin the centre of the cells increased 60 seconds after UV treatment,compared with before exposure. This indicates superoxide production.This initial increase in brightness faded as the ROS dye photo-bleaches.Imaging the ROS dye under the rhodamine filter induced photo-bleachingmuch more rapidly with the DC324 treated cells than with the othercompounds.

In order to gain more insight into the mode of action leading to celldeath, we examined the photo-initiated ROS production using the redoxreactive dye, CellRox, which fluoresces in response to oxidation byreactive oxygen species. DC324-treated cells stimulated with 405 nmlight exhibited a strong CellRox fluorescence signal after irradiation,particularly in intracellular organelles. CellRox fluorescence wasquantified in the cell before and after irradiation; a steady increasein the production of ROS-stimulated relative fluorescence was observedimmediately following irradiation in DC324-treated cells, but not cellstreated with EC23, a synthetic retinoid analogue of DC324 with ATRA-likebiochemical properties, which acted as a negative control, todemonstrate that co-treatment with any retinoid or near ATRA-likeanalogue and light does not kill cells.

Referring to FIG. 16 DC473 compound treated cells after 2 hours and 24hours UV exposure.

Referring to FIG. 17 a comparison is made of DC324 and DC473 compoundtreated cells.

Referring to FIG. 18 a comparison is made between the absorption spectraof DC324, DC473 and DC474 in CHCl₃.

Referring to FIG. 19 a comparison is made between the emission spectraof DC324, DC473 and DC474 in CHCl₃.

Referring to FIG. 20 a comparison is made between the absorption spectraof DC473, DC474 and click-conjugate compound 16 in CHCl₃.

Referring to FIG. 21 a comparison is made between the emission spectraof DC473, DC474 and click-conjugate compound 16 in CHCl₃.

REFERENCES

-   1 R. Comitato, T. Esposito, G. Cerbo, F. Angelini, B. Varriale    and A. Cardone, J. Exp. Zool. A. Comp. Exp. Biol., 2006, 305,    288-98.-   2 D.-D. Lee, O. Stojadinovic, A. Krzyzanowska, C. Vouthounis, M.    Blumenberg and M. Tomic-Canic, J. Cell. Physiol., 2009, 220, 427-39.-   3 M. Ethirajan, Y. Chen, P. Joshi and R. K. Pandey, Chem. Soc. Rev.,    2011, 40, 340-62.-   4 S. Grether-Beck, S. Olaizola-Horn, H. Schmitt, M. Grewe, A.    Jahnke, J. P. Johnson, K. Briviba, H. Sies and J. Krutmann, Proc.    Natl. Acad. Sci., 1996, 93, 14586-14591.-   5 I. Mfouo-Tynga and H. Abrahamse, Int. J. Mol. Sci., 2015, 16,    10228-41.-   6 Z. Wang, M. Boudjelal, S. Kang, J. J. Voorhees and G. J. Fisher,    Nat. Med., 1999, 5, 418-22.-   7 H.-U. Simon, A. Haj-Yehia and F. Levi-Schaffer, Apoptosis, 5,    415-418.-   8 Ryan F. Donnelly, Paul A. McCarron, and A. David Woolfson,    Perspect Medicin Chem. 2007; 1: 49-63. Published online 2007 Dec.    11; PMCID: PMC2754918 “Derivatives of 5-Aminolevulinic Acid for    Photodynamic Therapy”.-   9 Leanne B. Josefsen and Ross W. Boyle, Met Based Drugs. 2008; 2008:    276109; Published online 2008 Sep. 11; doi:10.1155/2008/276109;    PMCID: PMC2535827 “Photodynamic Therapy and the Development of    Metal-Based Photosensitisers”.

The invention claimed is:
 1. A method of treating a patient withphotodynamic therapy (PDT), the method comprising the administration ofa highly conjugated retinoid compound that generates reactive oxygenspecies when said compound is activated by light.
 2. The methodaccording to claim 1 wherein the highly conjugated retinoid compoundpossesses a retinoid structure that includes at least six conjugateddouble or triple bonds.
 3. The method according to claim 2 wherein thehighly conjugated retinoid compound is a compound of formula I:

in which A¹ is N or CR³; A² is N or CR⁴; A³ is N or CR⁵; A⁴ is N or CR⁶;R³, R⁴, R⁵ and R⁶, which may be the same or different, are eachhydrogen, alkyl_(C1-10), alkene_(C2-12), aryl, aralkyl, halogen,trifluoroalkyl, cyano, nitro, —NR^(a)R^(b), —OR^(a), glycol, —C(O)R^(a),—C(O)OR^(a), —OC(O)R^(a), —S(O)R^(a)R^(b), —C(O)NR^(a)R^(b) or asolubilising group; R¹ is —NR^(7a)R^(7b) or together with R⁶ forms aring II:

R⁷ and R^(7a) are each hydrogen, propynyl, —(CH₂)_(n)C≡CH, —(CH₂)_(n)SH,—(CH₂)SO₂F or —(CH₂)_(n)C═CH₂, alkyl_(C1-10), said alkyl beingoptionally substituted by aryl or heteroaryl; R^(7b) is hydrogen,propynyl, alkyl_(C1-10), said alkyl being optionally substituted by arylor heteroaryl; R⁸, R⁹, R¹⁰ and R¹¹, which may be the same or different,are each hydrogen or alkyl_(C1-4), aryl, halogen, trifluoroalkyl,—OR^(c) or glycol, or together one pair of R⁸ and R¹⁰ or R⁹ and R¹¹represent a bond; R¹² and R¹³, which may be the same or different, areeach hydrogen, alkyl_(C1-4) or together one pair of R¹⁰ and R¹² or R¹¹and R¹³ represent a bond, or R¹² and R¹³ together form a group:═CR¹⁴R¹⁵ provided that the pair of R¹⁰ and R¹² or R¹¹ and R¹³ does notrepresent a bond if a pair from R⁸, R⁹, R¹⁰ and R¹¹ represents a bond;R¹⁴ and R¹⁵, which may be the same or different, are each hydrogen oralkyl_(C1-10); and R^(a), R^(b) and R^(c), which may be the same ordifferent, are each hydrogen or alkyl_(C1-10); n is an integer from 1 to6; R² is a group III:

in which X^(a) is —C≡C—, —CH═CH— or N═CH—; X^(b) is —C≡C— or is absent;A⁵ is N or CR¹⁷; A⁶ is N or CR¹⁸; A⁷ is N or CR¹⁹; A⁸ is N or CR²⁰, R¹⁷,R¹⁸, R¹⁹ and R²⁰, which may be the same or different, are each hydrogen,alkyl_(C1-10), alkene_(C2-12), aryl, aralkyl, halogen, trifluoroalkyl,cyano, nitro, —NR^(d)R^(e), —OR^(d), glycol, —C(O)R^(d), —C(O)OR^(d),—OC(O)R^(d), —S(O)R^(d)R^(e), —C(O)NR^(d)R^(e) or a solubilising group;R¹⁶ is —CR²¹═CR²²Y, —C≡C—R²³ or together with R¹⁸ forms a ring IV:

A⁹ is N or CR²⁴; A¹⁰ is N or CR²⁵; A¹¹ is N or CR²⁶; R²³ is a group V:

in which A¹² is N or CR²⁷; A¹³ is N or CR²⁸; A¹⁴ is N or CR²⁹; A¹⁵ is Nor CR³⁰; R²¹ and R²², which may be the same or different, are eachhydrogen, alkyl_(C1-10), alkene_(C2-12), aryl, halogen ortrifluoroalkyl; R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹ and R³⁰, which may be thesame or different, are each hydrogen, alkyl_(C1-10), alkene-_(C2-12),aryl, halogen, trifluoroalkyl, —OR^(f), glycol or a solubilising group;R^(d), R^(e) and R^(f), which may be the same or different, are eachhydrogen or alkyl_(C1-10); Y is —CO₂R³¹, —COH, —CO₂CH₂C≡CH, —CN, —SF₅,—SO₃H, —SO₂NH₂, —SO₂CF₃, —CF₃, —CO₂(CH₂)_(m)SH, —CO₂(CH₂)_(m)SO₂F,—CO₂(CH₂)_(m)CH═CH₂, —C═NR³² or —C═N⁺R³³R³⁴; R³¹ is hydrogen,alkyl_(C1-10), alkene_(C2-12), aryl or a photocleavable group; R³², R³³and R³⁴, which may be the same or different, are each hydrogen,alkyl_(C1-10), alkene_(C2-12) or aryl; m is an integer from 1 to 9; andisomers thereof; in free or in salt form.
 4. The method according toclaim 3 wherein A¹ is CR³, A² is CR⁴, A³ is CR⁵ and A⁴ is CR⁶; and R¹,R², R³, R⁴, R⁵ and R⁶ are each as defined in claim
 3. 5. The methodaccording to claim 3 wherein A¹, A², A³, A⁴ and R² are each as definedin claim 3; and R¹ together with R⁶ forms a ring II:

wherein R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are each as defined in claim3.
 6. The method according to claim 5 wherein A¹ is CR³, A² is CR⁴, A³is CR⁵ and A⁴ is CR⁶.
 7. The method according to claim 3 wherein R² is agroup III:

wherein X^(a) is —C≡C— and X^(b) is —C≡C—; and A⁵, A⁶, A⁷, A⁸ and R¹⁶are each as defined in claim
 3. 8. The method according to claim 3wherein X^(b) is absent and X^(a) is selected from a group consistingof: —C≡C—, —CH═CH—, and —N═CH—.
 9. The method according to claim 3wherein A⁵ is CR¹⁷, A⁶ is CR¹⁸, A⁷ is CR¹⁹ and A¹⁸ is CR²⁰; and X^(a),X^(b), R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each as defined in claim
 3. 10.The method according to claim 3 wherein A⁵ is CR¹⁷, A⁶ is CR¹⁸, A⁷ isCR¹⁹ and A⁸ is CR²⁰; and R¹⁷, R¹⁹ and R²⁰ are each as defined in claim3; R¹⁶ together with R¹⁸ forms a ring IV:

wherein A⁹, A¹⁰, A¹¹ and Y are each as defined in claim
 3. 11. Themethod according to claim 3 wherein R² is a group III;

and R¹⁶ is —C≡C—R²³ wherein R²³ is a group V:

in which A¹² is CR²⁷, A¹³ is CR²⁸, A¹⁴ is CR²⁹ and A¹⁵ is CR³⁰; and R²⁷,R²⁸, R²⁹, R³⁰ and Y are each as defined in claim
 3. 12. The methodaccording to claim 11 wherein R¹⁶ is —C≡C—R²³, R²³ is a group V and Y is—CO₂R³¹, —COH, —CO₂CH₂C≡CH, —CN, —SF₅, —SO₃H, —SO₂NH₂, —SO₂CF₃, in whichR³¹ is hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl or a photocleavablegroup.
 13. The method according to claim 11 wherein Y is —CO₂R³¹ inwhich R³¹ is hydrogen, alkyl_(C1-10), alkene_(C2-12), aryl or aphotocleavable group.
 14. The method according to claim 3 wherein R⁷ orR^(7a) is alkyl C1-10.
 15. The method according to claim 3 wherein R⁸,R⁹, R¹⁰ and R¹¹ are each hydrogen.
 16. The method according to claim 3wherein R⁸ and R¹⁰ or R⁹ and R¹¹ represent a bond.
 17. The methodaccording to claim 3 wherein R¹² and R¹³ each represent alkyl C1-4. 18.The method according to claim 3 wherein R² is selected from a groupconsisting of: group VI:

group VII

group VIII

and group IX

wherein R³¹ is as defined in claim
 3. 19. The method according to claim3 wherein the compound is selected from the group consisting of:(2E)-3-(4-2-[4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoic acid;(2E)-3-(4-2-[4,4-dimethyl-1-(propyn-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoic acid; and Methyl(2E)-3-(4-{2-[4,4-dimethyl-1-(prop-2-yn-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl}phenyl)prop-2-enoate; and isomers thereof; in free or in salt form. 20.The method according to claim 3 wherein the highly conjugated retinoidcompound is comprised in a medicament for use in photodynamic therapy(PDT) is suitable for non-surgical cell ablation for the treatment ofone or more of cancer; the treatment of benign growths; the treatment ofimmune mediated inflammatory disorders; or the treatment of a diseasecaused by a pathogenic organism.
 21. The method according to claim 20wherein the medicament for use in photodynamic therapy (PDT) comprisesone or more additional therapeutic agents, wherein the one or moreadditional therapeutic agent may be selected from the group consistingof: a chemotherapeutic agent, an immunotherapeutic agent, a gene therapyagent, and a radiotherapeutic agent.
 22. The method according to claim20 wherein the medicament for use in photodynamic therapy (PDT) issuitable for the treatment of a disease caused by a pathogenic organism.23. The method according to claim 14 wherein R⁷ or R^(7a) is alkyl C1-3.24. The method according to claim 17 wherein R¹² and R¹³ each representmethyl.